draft - Thermo Fisher Scientific

draft - Thermo Fisher Scientific
Applied Biosystems
7900HT Fast Real-Time PCR System
and SDS Enterprise Database
User Guide
Applied Biosystems
7900HT Fast Real-Time PCR System
and SDS Enterprise Database
User Guide
DRAFT
November 19, 2007 11:09 am, 4351684A_Title.fm
© Copyright 2007, 2010 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
The Applied Biosystems 7900HT Fast Real-Time PCR System is a real-time thermal cycler covered by US patents and corresponding claims in their non-US counterparts, owned by Applied Biosystems. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus,
reagents, kits, or methods such as 5’ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing,
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
TRADEMARKS:
ABI PRISM, Applied Biosystems, MicroAmp, Primer Express, and VIC are registered trademarks and AB (Design), ABI PRISM, Applera, FAM, JOE, NED, ROX,
TAMRA, and TET are trademarks of Applied Biosystems or its subsidiaries in the US and/or certain other countries.
AmpErase, AmpliTaq Gold, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
SYBR is a registered trademark of Molecular Probes, Inc.
Zymark is a registered trademark of Zymark Corporation.
Windows and Windows NT are registered trademarks of Microsoft Corporation.
All other trademarks are the sole property of their respective owners.
Part Number 4351684 Rev. C
06/2010
DRAFT
November 19, 2007 11:09 am, 4351684A_Title.fm
Contents
Preface
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
How to Obtain Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
Safety and EMC Compliance Information
Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx
Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Bar Code Scanner Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii
Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii
Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . . . xxiii
Chapter 1
Product Overview
System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Section 1.1 Getting to Know the Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Instrument Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
Section 1.2 Getting to Know the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13
Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14
SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15
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Managing Sequence Detection System Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17
Section 1.3 SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-21
About the SDS Enterprise Database Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22
About the SDS Enterprise Database Software Suite . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25
Database Modules for Large-Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25
Database Management Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26
Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27
Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28
Chapter 2
Getting Started
Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Using the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 1: Using Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 2: Viewing and Resizing Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 3: Using the Plate Grid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 4: Using the Hand-Held Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 5: Using Contextual Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lesson 6: Using Keyboard Shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2-10
2-11
2-12
2-21
2-23
2-27
2-28
2-28
Using SDS Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29
Chapter 3
Preparing a Run
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Quick Review: Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
Step 2 – Applying Detectors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Step 3 – Configuring the Plate Document with Tasks . . . . . . . . . . . . . . . . . . . . . . . . . 3-14
Step 4 – Setting the Passive Reference and Omitting Wells . . . . . . . . . . . . . . . . . . . . 3-16
Step 5 – Programming the Plate Document Method . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17
Step 6 – Saving the Plate Document as a Template . . . . . . . . . . . . . . . . . . . . . . . . . . 3-22
Step 7 – Creating a Plate Document from the Template . . . . . . . . . . . . . . . . . . . . . . . 3-24
Step 8 – Applying Sample and Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-25
Step 9 – Running the Plate on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . 3-26
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Chapter 4
Operating the Instrument
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
Section 4.1 Consumable Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Preparing TaqMan Low Density Arrays for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the Centrifuge System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the Sealer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Loading the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Centrifuging the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sealing the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4-10
4-10
4-12
4-14
4-14
4-16
4-19
Section 4.2 Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23
Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24
Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-25
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29
Section 4.3 Automated Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-31
Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . 4-32
Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . . . .
Adding a Plate Document to the Plate Queue (Using the SDS Software) . . . . . .
Creating Plate Documents Using the Template Batch Utility . . . . . . . . . . . . . . . .
Starting and Configuring the Automation Controller Software for Operation . . . .
4-34
4-35
4-36
4-39
Running Plates Using the Automation Controller Software . . . . . . . . . . . . . . . . . . . . 4-41
Loading Plates onto the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-41
Running the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-43
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44
Chapter 5
Analyzing End-Point Data
End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3
Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11
Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12
Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18
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Chapter 6
Analyzing Real-Time Data
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10
Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11
Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14
Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16
Algorithmic Manipulation of Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17
Automatic Outlier Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19
Essential Experimental Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20
Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22
Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23
Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23
Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Specifying the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analyzing the Study Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6-26
6-26
6-29
6-30
Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35
Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Determining Tm Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44
Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45
Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46
Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
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Chapter 7
Maintaining the Instrument
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2
Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3
Section 7.1 Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-5
Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6
Changing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12
Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14
Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing the Background Plate or TaqMan Low Density Array . . . . . . . . . . . . .
Creating a Plate Document for the Background Run . . . . . . . . . . . . . . . . . . . . . .
Running the Prepared Background Plate or TaqMan Low Density Array . . . . . .
Analyzing the Background Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7-16
7-17
7-17
7-18
7-19
Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing the Pure Dye Plates or Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing a Plate Document for a Pure Dye Plate or Card . . . . . . . . . . . . . . . . .
Running the Prepared Pure Dye Plate or Card . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analyzing the Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7-20
7-22
7-22
7-24
7-25
Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27
Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30
Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35
Automation Accessory Components and Stack Positions . . . . . . . . . . . . . . . . . . . . . 7-36
Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . 7-37
Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41
Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49
Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52
Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . . . . . 7-53
General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54
Maintaining the SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55
Chapter 8
Troubleshooting
Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2
Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5
Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9
Pure Dye Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11
Real-Time Runs (Quantitative PCR and Dissociation Curves) . . . . . . . . . . . . . . . . . . 8-12
End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13
Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14
Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . . . . 8-17
TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18
SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20
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Appendix A Software Reference
Importing Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2
Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. File Version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Plate Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Plate ID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Detector Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Number of Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Detectors List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Detectors List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A-4
A-5
A-5
A-5
A-5
A-6
A-6
A-6
A-7
Marker Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Number of Markers (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . .
8. Markers List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . .
9. Markers List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A-8
A-8
A-8
A-9
Well-Detector Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10
10. Well-Detector List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10
11. Well-Detector Definition List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10
Well-Marker Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-12
12. Well-Marker List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . A-12
13. Well-Marker Definition List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . A-12
Example Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-14
Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16
Operating the SDS Software from a Command Line . . . . . . . . . . . . . . . . . . . . . . . . . . A-18
Using the Search Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-20
Connecting SDS Software to the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22
Appendix B Designing TaqMan Reagent-Based Assays
Assay Development Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2
Design Tips for Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5
Design Tips for Quantitative PCR Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6
Appendix C Kits, Reagents, and Consumables
Interchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . . . . . C-2
Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3
Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5
TaqMan Pre-Developed Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6
Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6
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Appendix D Theory of Operation
Fluorescent-Based Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Real-Time Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-4
Comparative CT Method of Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8
Appendix E Limited Warranty Statement
Bibliography
Glossary
Index
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Preface
How to Use This Guide
Purpose of This
Guide
The Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise
Database User Guide describes how to prepare, maintain, and troubleshoot the
Applied Biosystems 7900HT Fast Real-Time PCR System instrument, Zymark®
Twister Microplate Handler, and SDS Enterprise Database.
Audience
This guide is written for technicians, scientists, and researchers of all skill levels who
will use and maintain 7900HT instruments with or without the SDS Enterprise
Database.
Assumptions
This guide assumes that a Applied Biosystems technical representative has installed
your 7900HT instrument, Plate Handler, and SDS Enterprise Database and that you
are familiar with:
• Basic Microsoft® Windows® operations such as using the mouse, choosing
commands, working with windows, and using the hierarchical file system
• Electronic storage devices (computer hard drives) and data files
• Assay preparation and basic laboratory techniques
Text Conventions
This guide uses the following conventions:
• Bold indicates user action. For example:
Enter 0, then press Enter for each of the remaining fields.
• Italics indicates new or important words and is used for emphasis. For example:
Before performing a pure dye calibration, always perform a background run.
• A right arrow bracket (>) separates successive commands you select from a
drop-down or shortcut menu. For example:
Select File > Open > Spot Set.
User Attention
Words
Two user attention words appear in Applied Biosystems user documentation. Each
word implies a particular level of observation or action as described below:
Note: Provides information that may be of interest or help but is not critical to the
use of the product.
IMPORTANT! Provides information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
Safety Alert
Words
Safety alert words also appear in user documentation. For more information,
see “Safety Alert Words” on page xiv.
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Preface
How to Obtain More Information
Related
Documentation
See the following related documents for more information on the topics in this guide:
• Applied Biosystems 7900HT Fast Real-Time PCR System Quick Starts –
Provide brief, procedures for performing application-specific tasks on the
7900HT instrument.
• Sequence Detections Systems Software Online Help – Describes the Sequence
Detection Systems (SDS) Software and provides procedures for common tasks.
• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time
PCR System Administrators Guide – Provides information for database
administrators who will be maintaining the SDS Enterprise Database, and
provides information for systems integrators who will be working with the SDS
Enterprise Database API.
Note: For additional documentation, see “How to Obtain Services and Support” on
page xii.
Send Us Your
Comments
Applied Biosystems welcomes your comments and suggestions for improving its
user documents. You can e-mail your comments to:
[email protected]
How to Obtain Services and Support
To contact Applied Biosystems Technical Support from North America by telephone,
call 1.800.899.5858.
For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Services and Support.
At the Services and Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates of analysis,
and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Services and Support page provides access to worldwide telephone
and fax numbers to contact Applied Biosystems Technical Support and Sales
facilities.
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Safety and EMC Compliance Information
This section includes the following topics:
Safety Conventions Used in This Document. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xv
Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
General Instrument Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xx
Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Biological Hazard Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Laser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Bar Code Scanner Laser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii
Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii
Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . xxiii
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Safety and EMC Compliance Information
Safety Conventions Used in This Document
Safety Alert
Words
Four safety alert words appear in Applied Biosystems user documentation at points
in the document where you need to be aware of relevant hazards. Each alert
word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular
level of observation or action, as defined below:
Definitions
IMPORTANT! – Indicates information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
– Indicates a potentially hazardous situation that, if not avoided,
may result in minor or moderate injury. It may also be used to alert against unsafe
practices.
– Indicates a potentially hazardous situation that, if not avoided,
could result in death or serious injury.
– Indicates an imminently hazardous situation that, if not avoided,
will result in death or serious injury. This signal word is to be limited to the most
extreme situations.
Except for IMPORTANTs, each safety alert word in an Applied Biosystems
document appears with an open triangle figure that contains a hazard symbol. These
hazard symbols are identical to the hazard icons that are affixed to Applied
Biosystems instruments (see “Safety Symbols” on page xv).
Examples
The following examples show the use of safety alert words:
IMPORTANT! You must create a separate a Sample Entry Spreadsheet for each
96-well microtiter plate.
The lamp is extremely hot. Do not touch the lamp until it has
cooled to room temperature.
CHEMICAL HAZARD. Formamide. Exposure causes eye,
skin, and respiratory tract irritation. It is a possible developmental and birth defect
hazard. Read the MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
ELECTRICAL HAZARD. Failure to ground the instrument
properly can lead to an electrical shock. Ground the instrument according to the
provided instructions.
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Symbols on Instruments
Symbols on Instruments
Electrical
Symbols on
Instruments
The following table describes the electrical symbols that may be displayed on
Applied Biosystems instruments.
Symbol
Description
Symbol
Indicates the On position of the
main power switch.
Safety Symbols
Description
Indicates the Off position of the
main power switch.
Indicates a protective
grounding terminal that must
be connected to earth ground
before any other electrical
connections are made to the
instrument.
Indicates the On/Off position of
a push-push main power
switch.
Indicates a terminal that can
receive or supply alternating
current or voltage.
Indicates a terminal that may
be connected to the signal
ground reference of another
instrument. This is not a
protected ground terminal.
Indicates a terminal that can
receive or supply alternating or
direct current or voltage.
The following table describes the safety symbols that may be displayed on
Applied Biosystems instruments. Each symbol may appear by itself or in combination
with text that explains the relevant hazard (see “Safety Labels on Instruments” on
page xvi). These safety symbols may also appear next to DANGERS, WARNINGS,
and CAUTIONS that occur in the text of this and other product-support documents.
Symbol
Description
Symbol
Description
Indicates that you should
consult the manual for further
information and to proceed
with appropriate caution.
Indicates the presence of a
laser inside the instrument and
to proceed with appropriate
caution.
Indicates the presence of an
electrical shock hazard and to
proceed with appropriate
caution.
Indicates the presence of
moving parts and to proceed
with appropriate caution.
Indicates the presence of a hot
surface or other
high-temperature hazard and
to proceed with appropriate
caution.
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xv
Safety and EMC Compliance Information
Safety Labels on Instruments
The following CAUTION, WARNING, and DANGER statements may be displayed
on Applied Biosystems instruments in combination with the safety symbols
described in the preceding section.
English
Francais
CAUTION Hazardous chemicals. Read the Material Safety
Data Sheets (MSDSs) before handling.
ATTENTION Produits chimiques dangeureux. Lire les fiches
techniques de sûreté de matériels avant la manipulation des
produits.
CAUTION Hazardous waste. Read the waste profile (if any)
in the site preparation guide for this instrument before
handling or disposal.
ATTENTION Déchets dangereux. Lire les renseignements
sur les déchets avant de les manipuler ou de les éliminer.
CAUTION Hazardous waste. Refer to MSDS(s) and local
regulations for handling and disposal.
ATTENTION Déchets dangereux. Lire les fiches techniques
de sûreté de matériels et la régulation locale associées à la
manipulation et l'élimination des déchets.
WARNING Hot lamp.
AVERTISSEMENT Lampe brûlante.
WARNING Hot. Replace lamp with an Applied Biosystems
lamp.
AVERTISSEMENT Composants brûlants. Remplacer la
lampe par une lampe Applied Biosystems.
CAUTION Hot surface.
ATTENTION Surface brûlante.
DANGER High voltage.
DANGER Haute tension.
WARNING To reduce the chance of electrical shock, do not
remove covers that require tool access. No user-serviceable
parts are inside. Refer servicing to Applied Biosystems
qualified service personnel.
AVERTISSEMENT Pour éviter les risques d'électrocution, ne
pas retirer les capots dont l'ouverture nécessite l'utilisation
d'outils. L’instrument ne contient aucune pièce réparable par
l’utilisateur. Toute intervention doit être effectuée par le
personnel de service qualifié de Applied Biosystems.
DANGER Class 3B laser radiation present when open and
interlock defeated. Avoid direct exposure to laser beam.
DANGER Class 3B rayonnement laser en cas d’ouverture et
d’une neutralisation des dispositifs de sécurité. Eviter toute
exposition directe avec le faisceau.
DANGER Class 3B laser radiation when open. Avoid direct
exposure to laser beam.
DANGER Class 3B rayonnement laser en cas d’ouverture.
Eviter toute exposition directe avec le faisceau.
DANGER Class 3B laser radiation present when open and
interlock defeated. Do not stare directly into the beam
DANGER de Class 3B rayonnement laser en cas d'ouverture
et d'une neutralisation des dispositifs de securite. Eviter
toute exposition directe avec le faisceau.
DANGER Class 3B laser radiation present when open. Do
not stare directly into the beam.
DANGER de Class 3B rayonnement laser en cas
d'ouverture. Eviter toute exposition directe avec le faisceau.
DANGER Class 3B LED when open and interlock defeated.
Do not stare directly into the beam.
DANGER de Class 3B LED en cas d'ouverture et d'une
neutralisation des dispositifs de securite. Eviter toute
exposition directe avec le faisceau.
DANGER Class 3B LED when open. Do not stare directly
into the beam.
DANGER de Class 3B LED en cas d'ouverture. Eviter toute
exposition directe avec le faisceau.
CAUTION Moving parts.
ATTENTION Parties mobiles.
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General Instrument Safety
General Instrument Safety
PHYSICAL INJURY HAZARD. Use this product only as
specified in this document. Using this instrument in a manner not specified by
Applied Biosystems may result in personal injury or damage to the instrument.
Moving and
Lifting the
Instrument
PHYSICAL INJURY HAZARD. The instrument is to be moved
and positioned only by the personnel or vendor specified in the applicable site
preparation guide. If you decide to lift or move the instrument after it has been
installed, do not attempt to lift or move the instrument without the assistance of
others, the use of appropriate moving equipment, and proper lifting techniques.
Improper lifting can cause painful and permanent back injury. Depending on the
weight, moving or lifting an instrument may require two or more persons.
Moving and
Lifting StandAlone Computers
and Monitors
Do not attempt to lift or move the computer or the monitor
without the assistance of others. Depending on the weight of the computer and/or the
monitor, moving them may require two or more people.
Things to consider before lifting the computer and/or the monitor:
• Make sure that you have a secure, comfortable grip on the computer or the
monitor when lifting.
• Make sure that the path from where the object is to where it is being moved is
clear of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a good neutral position while lifting with your legs.
• Participants should coordinate lift and move intentions with each other before
lifting and carrying.
• Instead of lifting the object from the packing box, carefully tilt the box on its
side and hold it stationary while someone slides the contents out of the box.
Operating the
Instrument
Ensure that anyone who operates the instrument has:
• Received instructions in both general safety practices for laboratories and
specific safety practices for the instrument.
• Read and understood all applicable Material Safety Data Sheets (MSDSs). See
“About MSDSs” on page xviii.
PHYSICAL INJURY HAZARD. Use this instrument as
specified by Applied Biosystems. Using this instrument in a manner not specified by
Applied Biosystems may result in personal injury or damage to the instrument.
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Safety and EMC Compliance Information
Chemical Safety
Chemical Hazard
Warning
CHEMICAL HAZARD. Before handling any chemicals, refer to
the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all
relevant precautions.
CHEMICAL HAZARD. All chemicals in the instrument, including
liquid in the lines, are potentially hazardous. Always determine what chemicals have been
used in the instrument before changing reagents or instrument components. Wear
appropriate eyewear, protective clothing, and gloves when working on the instrument.
CHEMICAL HAZARD. Four-liter reagent and waste bottles can
crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety
container with the cover fastened and the handles locked in the upright position. Wear
appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.
CHEMICAL STORAGE HAZARD. Never collect or store waste
in a glass container because of the risk of breaking or shattering. Reagent and waste
bottles can crack and leak. Each waste bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in the
upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent
and waste bottles.
About MSDSs
Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with
shipments of hazardous chemicals to new customers. They also provide MSDSs with
the first shipment of a hazardous chemical to a customer after an MSDS has been
updated. MSDSs provide the safety information you need to store, handle, transport,
and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous chemical, be sure to
replace the appropriate MSDS in your files.
Obtaining
MSDSs
You can obtain from Applied Biosystems the MSDS for any chemical supplied by
Applied Biosystems. This service is free and available 24 hours a day.
To obtain MSDSs:
1. Go to www.appliedbiosystems.com, click Support, then click MSDS Search.
2. In the Keyword Search field, enter the chemical name, product name, MSDS
part number, or other information that appears in the MSDS of interest. Select
the language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of
the following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a
destination that you choose
Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact
the chemical manufacturer.
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Chemical Waste Safety
Chemical Safety
Guidelines
• Read and understand the Material Safety Data Sheets (MSDS) provided by the
chemical manufacturer before you store, handle, or work with any chemicals or
hazardous materials. (See “About MSDSs” on page xviii.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood). For additional
safety guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended on the MSDS.
• Comply with all local, state/provincial, or national laws and regulations related
to chemical storage, handling, and disposal.
Chemical Waste Safety
Chemical Waste
Hazard
HAZARDOUS WASTE. Refer to Material Safety Data Sheets and
local regulations for handling and disposal.
CHEMICAL WASTE HAZARD. Wastes produced by Applied
Biosystems instruments are potentially hazardous and can cause injury, illness, or death.
CHEMICAL STORAGE HAZARD. Never collect or store waste
in a glass container because of the risk of breaking or shattering. Reagent and waste
bottles can crack and leak. Each waste bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in the
upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent
and waste bottles.
Chemical Waste
Safety Guidelines
• Read and understand the Material Safety Data Sheets (MSDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle,
or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste
material and meet federal, state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).For additional
safety guidelines, consult the MSDS.
• Handle chemical wastes in a fume hood.
• After emptying the waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national environmental
and health regulations.
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xix
Safety and EMC Compliance Information
Waste Profiles
A waste profile for the Applied Biosystems 7900HT Fast Real-Time PCR System is
provided in the Applied Biosystems 7900HT Fast Real-Time PCR System Site
Preparation Guide.
Waste profiles show the percentage compositions of the reagents in the waste stream
generated during installation and during a typical user application, even though the
typical application may not be used in your laboratory.
The waste profiles help you plan for the handling and disposal of waste generated by
operation of the instrument. Read the waste profiles and all applicable MSDSs
before handling or disposing of chemical waste.
Waste Disposal
If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed
of according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special handling,
and disposal limitations may apply.
Electrical Safety
ELECTRICAL SHOCK HAZARD. Severe electrical shock can
result from operating the Applied Biosystems 7900HT Fast Real-Time PCR System
without its instrument panels in place. Do not remove instrument panels. Highvoltage contacts are exposed when instrument panels are removed from the
instrument.
Fuses
ELECTRICAL SHOCK HAZARD. Improper fuses or highvoltage supply can damage the instrument wiring system and cause a fire. Before
powering on the instrument, verify that the fuses are properly installed and that the
instrument voltage matches the power supply in your laboratory.
FIRE HAZARD. For continued protection against the risk of
fire, replace fuses only with fuses of the type and rating specified for the instrument.
Power
ELECTRICAL HAZARD. Grounding circuit continuity is vital
for the safe operation of equipment. Never operate equipment with the grounding
conductor disconnected.
ELECTRICAL HAZARD. Use properly configured and
approved line cords for the voltage supply in your facility.
ELECTRICAL HAZARD. Plug the system into a properly
grounded receptacle with adequate current capacity.
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Physical Hazard Safety
Overvoltage
Rating
The Applied Biosystems 7900HT Fast Real-Time PCR System has an installation
(overvoltage) category of II, and is classified as portable equipment
Physical Hazard Safety
Moving Parts
PHYSICAL INJURY HAZARD. Moving parts can crush and
cut. Keep hands clear of moving parts while operating the instrument. Disconnect
power before servicing the instrument.
PHYSICAL INJURY HAZARD. Do not operate the instrument
without the arm shield in place. Keep hands out of the deck area when the instrument
is spotting.
Biological Hazard Safety
General
Biohazard
BIOHAZARD. Biological samples such as tissues, body fluids,
and blood of humans and other animals have the potential to transmit infectious
diseases. Follow all applicable local, state/provincial, and/or national regulations.
Wear appropriate protective eyewear, clothing, and gloves. Read and follow the
guidelines in these publications:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories (stock no. 017-04000547-4; http://bmbl.od.nih.gov)
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030; http://www.access.gpo.gov/nara/cfr/
waisidx_01/29cfr1910a_01.html).
Additional information about biohazard guidelines is available at:
http://www.cdc.gov
Laser Safety
Laser
Classification
The Applied Biosystems 7900HT Fast Real-Time PCR System uses a Class 3B laser.
Under normal operating conditions, the instrument laser is categorized as a Class 3B
laser. When safety interlocks are disabled during certain servicing procedures, the
laser can cause permanent eye damage, and, therefore, is classified under those
conditions as a Class 3B laser.
The Applied Biosystems 7900HT Fast Real-Time PCR System complies with 21
CFR, 1040.10 and 1040.11, as applicable.
The Applied Biosystems 7900HT Fast Real-Time PCR System has been tested to and
complies with the “Radiation Control for Health and Safety Act of 1968 Performance
Standard CFR 1040.”
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xxi
Safety and EMC Compliance Information
The Applied Biosystems 7900HT Fast Real-Time PCR System has been tested to and
complies with standard EN60825-1, “Radiation Safety of Laser Products, Equipment
Classification, Requirements, and User’s Guide.”
Laser Safety
Requirements
Additional Laser
Safety
Information
To ensure safe laser operation:
• The system must be installed and maintained by an Applied Biosystems
Technical Representative.
• All instrument panels must be in place on the instrument while the instrument is
operating. When all panels are installed, there is no detectable radiation present.
If any panel is removed when the laser is operating (during service with safety
interlocks disabled), you may be exposed to laser emissions in excess of the
Class 3B rating.
• Do not remove safety labels or disable safety interlocks.
Refer to the user documentation provided with the laser for additional information on
government and industry safety regulations.
LASER HAZARD. Lasers can burn the retina causing
permanent blind spots. Never look directly into the laser beam. Remove jewelry and
other items that can reflect the beam into your eyes. Do not remove the instrument
top or front panels. Wear proper eye protection and post a laser warning sign at the
entrance to the laboratory if the top or front panels are removed for service.
LASER BURN HAZARD. An overheated laser can cause
severe burns if it comes in contact with the skin. DO NOT operate the laser when it
cannot be cooled by its cooling fan. Always wear appropriate laser safety goggles.
Bar Code Scanner Laser Safety
Laser
Classification
The bar code scanner included with the Applied Biosystems 7900HT Fast Real-Time
PCR System is categorized as a Class 2 (II) laser.
Laser Safety
Requirements
Class 2 (II) lasers are low-power, visible-light lasers that can damage the eyes.
Never look directly into the laser beam. The scanner is designed to prevent human
access to harmful levels of laser light during normal operation, user maintenance, or
during prescribed service operations.
LASER HAZARD. Class 2 (II) lasers can cause damage to eyes.
Avoid looking into a Class 2 (II) laser beam or pointing a Class 2 (II) laser beam into
another person’s eyes.
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Workstation Safety
Workstation Safety
Correct ergonomic configuration of your workstation can reduce or prevent effects
such as fatigue, pain, and strain. Minimize or eliminate these effects by configuring
your workstation to promote neutral or relaxed working positions.
MUSCULOSKELETAL AND REPETITIVE MOTION
HAZARD. These hazards are caused by potential risk factors that include but are not
limited to repetitive motion, awkward posture, forceful exertion, holding static
unhealthy positions, contact pressure, and other workstation environmental factors.
To minimize musculoskeletal and repetitive motion risks:
• Use equipment that comfortably supports you in neutral working positions and
allows adequate accessibility to the keyboard, monitor, and mouse.
• Position the keyboard, mouse, and monitor to promote relaxed body and head
postures.
Safety and Electromagnetic Compatibility (EMC) Standards
This section provides information on:
•
•
•
•
U.S. and
Canadian Safety
Standards
U.S. and Canadian Safety Standards
Canadian EMC Standard
European Safety and EMC Standards
Australian EMC Standards
This instrument has been tested to and complies with standard UL 3101-1, “Safety
Requirements for Electrical Equipment for Laboratory Use, Part 1: General
Requirements.”
This instrument has been tested to and complies with standard CSA 1010.1, “Safety
Requirements for Electrical Equipment for Measurement, Control, and Laboratory
Use, Part 1: General Requirements.”
Canadian EMC
Standard
European Safety
and EMC
Standards
This instrument has been tested to and complies with ICES-001, Issue 3: Industrial,
Scientific, and Medical Radio Frequency Generators.
Safety
This instrument meets European requirements for safety (Low Voltage Directive
73/23/EEC). This instrument has been tested to and complies with standards
EN 61010-1:2001, “Safety Requirements for Electrical Equipment for Measurement,
Control and Laboratory Use, Part 1: General Requirements” and EN 61010-2-010,
“Particular Requirements for Laboratory Equipment for the Heating of Materials.”
EMC
This instrument meets European requirements for emission and immunity (EMC
Directive 89/336/EEC). This instrument has been tested to and complies with
standard EN 61326 (Group 1, Class B), “Electrical Equipment for Measurement,
Control and Laboratory Use – EMC Requirements.”
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xxiii
Safety and EMC Compliance Information
Australian EMC
Standards
xxiv
This instrument has been tested to and complies with standard AS/NZS 2064,
“Limits and Methods Measurement of Electromagnetic Disturbance Characteristics
of Industrial, Scientific, and Medical (ISM) Radio-frequency Equipment.”
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Product Overview
In This Chapter
1
1
System Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Section 1.1 Getting to Know the Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Instrument Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
Section 1.2 Getting to Know the Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13
Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14
SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15
Managing Sequence Detection System Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17
Section 1.3 SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-21
About the SDS Enterprise Database Feature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22
About the SDS Enterprise Database Software Suite . . . . . . . . . . . . . . . . . . . . . . . 1-25
Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27
Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28
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1-1
Chapter 1 Product Overview
System Overview
About the
7900HT
Instrument
The Applied Biosystems 7900HT Fast Real-Time PCR System is a
second-generation sequence detection system instrument designed for automated,
high-throughput detection of fluorescent PCR-related chemistries. The instrument is
capable of real-time, end-point, and dissociation curve analysis of assays arrayed on
multiple formats. The 7900HT instrument is optimized for use with
Applied Biosystems chemistries for nucleic acid quantification and detection.
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Supported Runs
and Chemistries
The Applied Biosystems 7900HT Fast Real-Time PCR System provides two types of
runs to support the variety of PCR-related chemistries available from
Applied Biosystems and affiliated companies.
End-Point (Plate Read) Chemistry
In an end-point run, the thermal cycling of prepared plates containing reagents and
template is performed on a dedicated thermal cycler. Following the PCR, the 7900HT
instrument is then used to collect a plate-read or a single reading of the fluorescence
resulting from the completed reactions. Allelic Discrimination is an example of an
end-point chemistry that is directly supported by the SDS software.
Real-Time Chemistry
In a real-time run, the thermal cycling of prepared plates containing reagents and
template is performed on the 7900HT instrument. During the run, the instrument
collects data at each cycle of the PCR. The resulting data provides a chronological
series of measurements of the fluorescence resulting from the reactions. Examples of
real-time chemistry include absolute quantification, relative quantification and
dissociation curve analysis.
1-2
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Section 1.1 Getting to Know the Hardware
Section 1.1 Getting to Know the Hardware
In This Section
Instrument
Components
7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Instrument Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
The Applied Biosystems 7900HT Fast Real-Time PCR System consists of the
components shown in Figure 1-2.
Microsoft® Windows®
Compatible Computer
(see page 1-6)
7900HT Instrument
(see page 1-4)
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-Position
Bar Code Reader
(see page 1-7)
Figure 1-2
System
Zymark® Twister
Microplate Handler
(see page 1-8)
Extended
Capacity Stacks
(see page 1-7)
Hand-Held Bar
Code Reader
(see page 1-7)
Components of the Applied Biosystems 7900HT Fast Real-Time PCR
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1-3
Chapter 1 Product Overview
7900HT Instrument
Internal
Components
The 7900HT instrument contains the hardware used for thermal cycling and
detection of fluorescent chemistries (see page 1-2). Figure 1-3 illustrates the major
subcomponents of the instrument described in detail below.
Do not remove the cover to the Applied Biosystems 7900HT Fast
Real-Time PCR System. Only a qualified Applied Biosystems service engineer may
repair or adjust the internal components of the 7900HT instrument. Failure to comply
can result in serious injury and/or damage to the instrument.
Laser
CCD
camera
Optics
system
Reader
Heated clamp
GR2112
Tray
Door
Automated plate
handling system
Sample block module
Figure 1-3
Automated Plate
Handling System
Internal Components of the 7900HT Instrument
PHYSICAL HAZARD. Keep hands and loose clothing away
from the instrument tray and door at all times during instrument operation. The
7900HT instrument contains several internal components that can cause serious
physical injury.
The 7900HT instrument features an automated plate-handling system to provide easy
loading and removal of plates from the instrument. In combination with the
automation module, the plate-handling system allows unattended operation of the
instrument.
Heated Clamp
PHYSICAL INJURY HAZARD. Hot Surface. Use care when
working around this area to avoid being burned by hot components.
The 7900HT instrument has a heated clamp that provides optimal heat transfer and
uniform heating during thermal cycling. When the instrument tray loads a plate, the
clamp applies a downward pressure of 70 lbs (31.8 kg) onto the consumable. During
the run, the clamp maintains a constant temperature of 105 °C (±3 °C) to prevent
condensation within the consumable wells.
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7900HT Instrument
Interchangeable
Thermal Cycler
Blocks
The 7900HT instrument features a Peltier-based, interchangeable sample block
module based on the technology established in the GeneAmp® PCR System 9700
thermal cycler. The sample block module houses an internal Peltier heating/cooling
unit (Figure 1-4). The sample block module is made of aluminum to provide an
optimal thermal transfer rate between the block and the optical plate or the
TaqMan® Low Density Array.
PHYSICAL INJURY HAZARD. Hot Surface. Use care when
working around this area to avoid being burned by hot components.
Connection to the instrument
(Do Not Touch)
GR2027
Warning
GR2028
GR2028
Circuitry
(Do Not Touch)
Block inside
instrument
Figure 1-4
Sample block
(Do Not Touch)
Heat sinks
(Do Not Touch)
Top view
Bottom view
Components of the Interchangeable Thermal Cycler Block
The interchangeable sample block module provides:
Wide temperature range: 4–99.9 °C
Accuracy: ±0.25 °C from 35–99.9 °C
Heat/cool rate: 1.5 °C per second
Temperature uniformity: ±0.5 °C (measured 30 sec after the clock starts)
Long-term stability and high reliability
Support for multiple consumable formats (see page 1-9)
Several different modes of operation including 9600 mode and programmable
temperature ramps (see page 3-19)
• Reduced instrument downtime by allowing immediate replacement of the block
(see page 7-6)
• Easy access to the sample block for troubleshooting and maintenance
(see page 7-6)
•
•
•
•
•
•
•
Optics System
IMPORTANT! Do not remove the cover to the 7900HT instrument. Only a qualified
Applied Biosystems service engineer may repair or adjust the internal components.
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1-5
Chapter 1 Product Overview
Computer
Functions
A Microsoft Windows-compatible computer is required to operate the Applied
Biosystems 7900HT Fast Real-Time PCR System and the Zymark Twister
Microplate Handler. Through the SDS and Automation Controller Software, the
computer:
• Provides the interface for programming and operating the instrument
• Coordinates the operation of the 7900HT instrument, automation module, and
the bar code readers
• Provides storage and management of the data produced by the 7900HT
instrument
• Provides the local area network (LAN) connection to the SDS Enterprise
Database (if available)
System
Requirements
The system requirements for the computer used to operate the 7900HT instrument
vary with the version of the SDS software. To determine the system requirements for
your instrument, check the release notes accompanying your version of the SDS
software.
The release notes for the SDS software are in the following location:
D:\AppliedBiosystems\SDS2.2.1\ReleaseNotes.txt
Note: You can use the Microsoft Notepad, Wordpad, or Word software to read the
release notes text file.
Hard Drive
Partitions
During installation of the 7900HT instrument, the Applied Biosystems service engineer
partitions the computer hard drive to create the logical drives shown in the table below.
Primary Hard Drive
Drive
Size
(GB)
C
2
D
≥25
Contains
Operating system files*
•
•
•
•
•
•
•
SDS Software
Automation Controller Software
Zymark Twister Software
LAVA Software†
Additional Third-Party Software
SDS 7900HT Documents
(Optional) SDS Enterprise
Database Client Software
*Applied Biosystems recommends that you do not install programs to the C drive. The
computer will boot faster if the C drive contains only the operating system.
†Software required for the operation of the bar code reader.
Note: The partitions shown above represent the basic drive architecture required by
the SDS software. The actual number and size of the partitions and drives available
on your computer vary depending on the model of computer accompanying your
instrument.
1-6
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Bar Code Readers
Bar Code Readers
Description
The Applied Biosystems 7900HT Fast Real-Time PCR System includes two bar code
readers for data entry and plate recognition:
• A hand-held bar code reader for scanning plates manually
• A fixed-position bar code reader for automatically scanning plates as they are
loaded into the instrument (available only with the Automation Accessory)
Both bar code readers use a 670 nm Class II laser to scan plates and are capable of
reading Code 128 (alphanumeric), which supports 128 ASCII character bar codes.
Locations of the
Bar Code
Readers
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-Position Bar Code Reader
Hand-Held Bar Code Reader
Splitter
To computer
keyboard port
GR2018
~~
~
~
To keyboard
GR2015
~
~
(Shown with cover removed)
Figure 1-5
Using the
Bar Code
Readers
Locations of the Bar Code Readers of the 7900HT Instrument
LASER HAZARD. Exposure to direct or reflected laser light
can burn the retina and leave permanent blind spots. Never look into the laser beam.
Remove jewelry and anything else that can reflect the beam into your eyes. Protect
others from exposure to the beam.
For more information on the bar code readers, see:
Using the Hand-Held Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-27
Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49
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1-7
Chapter 1 Product Overview
Zymark Twister Microplate Handler
Description
The Zymark® Twister Microplate Handler coordinates plate handling for the 7900HT
instrument, permitting 24-hour unattended operation. The arm provides a 310-degree
rotational swing that permits access to the 7900HT instrument drawer, up to five
plate stacking areas, and the fixed-position bar code reader.
Zymark Twister
Microplate
Handler
Components
Zymark Twister
Microplate Handler
(cross-sectional view of the gripper)
Plate-sensor Gripper
switch
Fixed-position
bar code reader
Figure 1-6
Expansion
stacks
Adjustment
knob
Plate stack
Components of the Zymark Twister Microplate Handler
Plate Positions
X
4
3
1
2
Bar code
Zymark Twister Microplate Handler
(top view)
Figure 1-7
Using the
Zymark Twister
Microplate
Handler
Well A1
Plate Positions of the Zymark Twister Microplate Handler
LASER HAZARD. Exposure to direct or reflected laser light
can burn the retina and leave permanent blind spots. Never look into the laser beam.
Remove jewelry and anything else that can reflect the beam into your eyes. Protect
others from exposure to the beam.
For information on the Zymark Twister Microplate Handler, see:
Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4
Starting and Configuring the Automation Controller Software for Operation . . .4-39
Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-35
1-8
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Compatible Consumables
Compatible Consumables
Consumables for
Use with the
7900HT
Instrument
The interchangeable sample block modules of the 7900HT instrument allow it to
support a variety of consumable formats listed in Table 1-1.
Table 1-1
Compatible Consumables
Consumable
Compression
Pads for the
ABI PRISM
96-Well Optical
Reaction Plates
Compatible Seals
ABI PRISM™ 384-Well Optical
Reaction Plates
• ABI PRISM™ Optical Adhesive Covers
(quantity 100)
• Optical Adhesive Covers (quantity 25)
ABI PRISM™ 96-Well Optical
Reaction Plates
• Compression Pads:
– Standard pad for manual operation
– ABI PRISM™ Snap-On Compression Pad
(reusable)
• 96-Well Plate Seals:
– ABI PRISM™ Optical Adhesive Covers
(quantity 100)
– Optical Adhesive Covers (quantity 25)
– ABI PRISM™ Optical Caps (flat caps only)
Optical 96-Well Fast Thermal
Cycling Plates
• ABI PRISM™ Optical Adhesive Covers
(quantity 100)
• Optical Adhesive Covers (quantity 25)
TaqMan® Low Density Arrays
None (self sealing)
To ensure optimal heat transfer, Applied Biosystems recommends using compression
pads when running the ABI PRISM 96-Well Optical Reaction Plates.
If you are going to run the plate:
• Individually using the SDS software, then apply a standard compression pad
• As part of a batch using the Automation Controller Software, then apply an
ABI PRISM Snap-On Compression Pad
IMPORTANT! The ABI PRISM™ Snap-On Compression Pad/plate assembly may still
be hot after the Zymark® Twister Microplate Handler loads it into the Plate Handler’s
output stack. Please wait at least 30 seconds before manually handling the Snap-On
Compression Pad/plate assembly.
IMPORTANT! Performance of the Optical 96-Well Fast Thermal Cycling Plates with
the Zymark Twister Microplate Handler has not been verified. Therefore, when
running multiple Optical 96-Well Fast Plates on the 7900HT instrument, Applied
Biosystems recommends that you load and unload the plates manually.
Consumable
Requirements
See Appendix C, “Kits, Reagents, and Consumables,” for a list of available
consumables, requirements, and purchasing instructions.
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1-9
Chapter 1 Product Overview
Instrument Connections
Electrical
Connections
Figure 1-8 and Table 1-2 illustrate the electrical connections of the Applied
Biosystems 7900HT Fast Real-Time PCR System components.
Power
A
H
HI-POT
A
Power
B
C
D
GR2020
Power
HI-POT
A
B
C
E
A
G
F
B
Power
C
D
E
G
Power
C
Communications Cable
Power Cable
D
Figure 1-8
System
Connections of the Applied Biosystems 7900HT Fast Real-Time PCR
Table 1-2
System
Connections of the Applied Biosystems 7900HT Fast Real-Time PCR
Cable
Type
Connects…
To…
A
Communication
Computer (Monitor Port)
Monitor
B
Comm/Power
Computer (Mouse Port)
Mouse (not shown)
C
Serial
Computer (Serial Port 1)
7900HT Instrument
D
Comm/Power
Computer (Keyboard Port)
Hand-held Bar Code Reader
E
Communication
Computer (Serial Port 2)
Plate Handler (Port C)
F
Ethernet
Network
Computer (Ethernet Port)
G*
Comm/Power
Computer (ISA Card 1)
Fixed-Position Bar Code Reader
H
Comm/Power
Bar Code Reader Cable
Keyboard (not shown)
*See Figure 1-9 on page 1-11.
1-10
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Instrument Connections
Fixed-Position
Bar Code Reader
To Lava Card
(ISA Card 1)
Bar Code
Reader Cable
Power supply
Power
G
Figure 1-9
Fixed-Position Bar Code Reader Connection
Universal Voltage
Accessory Kit
(P/N 4334482)
IEC 320 4-Position
Universal Power Strip
(PN 4333969)
IEC 320 North America × (1)
Universal Jumper Cord set × (4)
Jumper Cord set for Japan × (4)
(see below for use in Japan)
IEC 320 Continental
IEC 320 U.K./Ireland × (1)
To computer, monitor, hand-held bar
code reader, and the Plate Handler
IEC 320 Australia/
New Zealand × (1)
Note: The order is not critical.
IEC 320 Japan × (1)
In Bag Marked “JAPAN ONLY”
Figure 1-10
North America: [email protected] MAX
Europe and all other locations: [email protected] MAX
Universal Power Strip
To install the Universal Voltage Accessory Kit (All Countries Except Japan):
1. Connect one universal jumper to each accessory (see below for use in Japan).
2. Connect the other end of the power jumpers to the power strip outputs.
3. Choose the correct country specific power input cord for the geographical region
and connect it to the power strip input.
4. Power off all instrument accessories (monitor, computer, and Plate Handler).
5. Connect the power-input cord to the AC outlet.
6. Power on accessories.
7. Discard unused cord sets.
Usage Guidelines for Japan
1. Use only the cord sets supplied in the bag marked “JAPAN ONLY.”
2. Discard all other unused cord sets.
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Chapter 1 Product Overview
1-12
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Section 1.2 Getting to Know the Software
Section 1.2 Getting to Know the Software
In This Section
Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14
SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15
Managing Sequence Detection System Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17
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1-13
Chapter 1 Product Overview
Software Components
Components of
the SDS Software
Suite
The Applied Biosystems 7900HT Fast Real-Time PCR System uses a suite of
applications to set up, run, and analyze experiments completed on the 7900HT
instrument.
Table 1-3
Standard SDS Software
Software
Function
SDS Software
• Constructs and edits plate document files
• Performs initial and end analysis of raw data from
allelic discrimination, relative quantification, and
absolute quantification runs
• Saves, prints, and exports run data
Java™ Runtime Environment
Additional files and software used to run the SDS
software
IMPORTANT! Do not delete the Java Runtime
Environment files. These files are crucial to the operation
of the SDS software.
7900HT Instrument Firmware
Table 1-4
• Controls the most basic operations of the 7900HT
instrument
• Controlled by commands sent from the computer
• Links the commands issued by the SDS software and
the operation of the 7900HT instrument
Software for Automated or SDS Enterprise Database Operation
Software
Function
Automation Controller
Software
• Controls and coordinates the action of the 7900HT
instrument and the automation module
• Initiates and controls the sequence detection run
• Acquires data during the run
Zymark Twister Software
Used to calibrate the Zymark Twister Microplate Handler.
LAVA Software
Used to align the fixed-position bar code reader.
SDS Enterprise Database
Client Software
Links the SDS software to an SDS Enterprise Database
on the network.
Note: See page 1-21 for more information on the
software components of the SDS Enterprise Database.
1-14
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SDS Software Related Files and Formats
SDS Software Related Files and Formats
Files Used and
Created by the
SDS Software
IMPORTANT! Never move or delete program files or directories for SDS-related
software unless specifically directed to do so by an Applied Biosystems
representative or documentation.
The SDS software uses many different files and directories (Table 1-5). Some of
these files store run data and calibration data: others are required to run the 7900HT
instrument.
Table 1-5
Common SDS Files
File Type
Ext.
Icon
Description/Comments
Plate Document Files
SDS 7900HT
Document
*.sds
• Serves as a virtual representation of a single
consumable used to contain samples and
reagents during a sequence detection run
• Contains all sample and assay data (names,
locations, reporter dye) contained by the
consumable
• Contains instructions for running the instrument
(thermal cycling and data collection parameters)
• Stores data gathered during the run
SDS 7900HT
Multiple Plate
Document
*.sdm
• Used to analyze relative quantification data
(small studies)
SDS 7900HT
Template
Document*
*.sdt
• Used to create plate documents
• Can store assay data, thermal cycling, and data
collection parameters
Imported/Exported Files
Assay Plate
Document
Setup Files
*.txt
Used to create plate documents
Exported Data
Files
*.txt
• Contains exported raw or analyzed data for all or
a select group of wells on a plate document
• Uses (*.txt) a tab-delimited format (compatible
with most spreadsheet applications)
JPEG† Graphic
File
*.jpg
‡
• Most panes and plots of the plate document can
be exported as JPEG graphic files
• Compatible with most word processing and
spreadsheet applications
• Can be incorporated directly into HTML or XML
documents
*The use of plate document templates is optional but useful as a timesaving device for
experiments where samples are run on plates with identical assay configurations.
†Joint Photographic Experts Group
‡Icon will varies depending on the application associated with the image file type
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1-15
Chapter 1 Product Overview
Plate Document
File Size
The SDS software can produce plate document files of varying sizes, depending on
the type of runs for which they are created. Table 1-6 lists the average sizes of typical
files produced by the SDS software.
Table 1-6
Average Plate Document File Sizes
File Size*
Run Type
Application
Plate-Read
Allelic Discrimination
Real-Time†
Absolute Quantification
Uncompressed
Compressed
150 to 180 KB
70 to 90 KB
15 to 25 MB‡
10 to 15 MB
Relative Quantification
*Compressed file sizes are estimates based on standard compression using the WinZip®
utility. For more information, see “Archiving SDS Files” on page 7-54.
†The maximum file sizes displayed above are nominal for real-time runs (absolute or relative
quantification). File size can increase depending on the plate document’s data collection
options.
‡The file size for a real-time run depends on the length of the run and the configuration of the
data collection options in the plate document. Longer runs and runs configured to collect
data at multiple stages of the method can be considerably larger than the 15 to 25 MB
average.
IMPORTANT! Because of memory constraints, the SDS Enterprise Database cannot
save real-time plate documents greater than 40 MB.
1-16
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Managing Sequence Detection System Data
Managing Sequence Detection System Data
System
Operation and
Dataflow
Standard Modes
of Operation
Data management strategy is a crucial element of successfully integrating the
Applied Biosystems 7900HT Fast Real-Time PCR System into a laboratory
workflow. During a single 24-hour period of real-time operation, the 7900HT
instrument can produce more than 200 MB of data. To successfully manage the
information produced by the 7900HT instrument, you should have a basic
understanding of how data is collected, stored, and processed throughout the
operation of the instrument.
The Applied Biosystems 7900HT Fast Real-Time PCR System has three standard
modes of operation depending on the accessories purchased with the base instrument
• Stand-Alone Operation (see below)
• Automated Operation (see page 1-18)
• Database Operation (see page 1-18)
Stand-Alone Operation
This mode uses the most basic configuration of the Applied Biosystems 7900HT Fast
Real-Time PCR System (Figure 1-11) where the 7900HT instrument is used without any
additional components. In this configuration, a technician runs plates individually using
the SDS software which stores all data on the computer hard drives.
Computer
SDS Software
GR2179
7900HT raven
instrument front
1
7900HT FAST Real-Time PCR System
Plate Document
Created
Applied Biosystems 7900HT
Fast Real-Time PCR System
Hard drive(s)
2
Plate Document Used
to Run a Plate
Instrument Firmware
3
5
Thermal Cycling and
Sequence Detection
Raw Data Saved to
the Plate Document
4
*.sds
file
*.sdt
file
6
Data Collection
Figure 1-11
Serial
Cable
Plate Document
Opened and Analyzed
Stand-Alone Operation of the 7900HT Instrument
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Chapter 1 Product Overview
Automated Operation
In this mode, the Applied Biosystems 7900HT Fast Real-Time PCR System uses an
Automation Accessory (Zymark Twister Microplate Handler and fixed-position bar
code reader), to provide high-throughput operation suitable for small- to
medium-scale studies. With this configuration (Figure 1-12), a technician can run
batches of plates unattended using the Automation Controller Software. As in
Stand-Alone operation, the computer stores all data for operation of the instrument
and provides the software for analyzing the run data.
Computer
SDS Software
1
Plate Documents
Created
Automation Controller
Software
GR2009
7900HT
Front view with Robot
2
Hard drive(s)
7900HT FAST Real-Time PCR System
Plate document files
added to and run from
the plate queue
GR2009
Applied Biosystems 7900HT
Fast Real-Time PCR System
*.sds
files
5
Instrument Firmware
Raw Data Saved to
the Plate Documents
3
*.sdt
file
Thermal Cycling and
Sequence Detection
4
6
Data Collection
Serial
Cable
Figure 1-12
Plate Document
Opened and Analyzed
Automated Operation of the of the 7900HT Instrument
Database Operation
For this mode, the Applied Biosystems 7900HT Fast Real-Time PCR System uses
the SDS Enterprise Database and the Automation Accessory, to provide maximum
high-throughput potential. In this configuration (Figure 1-13 on page 1-19), the
7900HT instrument has the storage capacity and analysis capability to support
medium- to large-scale gene expression and genotyping studies. Moreover, the SDS
Enterprise Database functions as a repository for all SDS-related data produced by
the networked client computers used for data collection and analysis.
1-18
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Managing Sequence Detection System Data
Data Collection Client
(Desktop Computer)
Applied Biosystems 7900HT
Fast Real-Time PCR System
Automation Controller
Software
Instrument Firmware
2
3
Plate documents run
from the database
Thermal Cycling and
Sequence Detection
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6
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6
Analysis Sessions
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Figure 1-13 Automated Operation of the of the 7900HT Instrument with
SDS Enterprise Database
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1-19
Chapter 1 Product Overview
1-20
DRAFT
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
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Section 1.3 SDS Enterprise Database
Section 1.3 SDS Enterprise Database
In This Section
About the SDS Enterprise Database Feature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22
About the SDS Enterprise Database Software Suite . . . . . . . . . . . . . . . . . . . . . . . 1-25
Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27
Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28
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1-21
Chapter 1 Product Overview
About the SDS Enterprise Database Feature
About the
Database
The SDS software can interface directly with an Applied Biosystems SDS Enterprise
Database as part of a laboratory information management system (LIMS). The SDS
Enterprise Database is a relational, Oracle-based repository designed specifically to
store SDS data produced by an Applied Biosystems 7900HT Fast Real-Time PCR
System. In addition to serving as a common repository for SDS data, the database
provides user authentication, robust and scalable data management, and flexible
archive capabilities. The database also provides the infrastructure and tools necessary
to integrate a 7900HT instrument into a laboratory workflow made up of existing
data management solutions.
Access Control
The SDS Enterprise Database provides a multi-level user security system that
restricts access to the database and to most functions of the SDS software and
7900HT instrument. The database provides the SDS User Account Manager software
with the client version of the SDS software for managing user accounts. The User
Manager allows users with Administrator privileges to assign privileges and
passwords for user accounts, and to add and remove them. This guide indicates the
access privileges required to perform each task using a short note above each
procedure.
Note: See the SDS Enterprise Database for the Applied Biosystems 7900HT Fast
Real-Time PCR System Administrators Guide (PN 4351669) for a complete
description of user accounts for the SDS Enterprise Database.
Network
Architecture
The SDS Enterprise Database is a relational, Oracle-based repository designed to store
SDS data produced by Applied Biosystems 7900HT Fast Real-Time PCR System
instruments. In addition to serving as a common repository for SDS data, the database
provides user authentication, robust and scalable data management, and flexible archive
capabilities. The database also provides the infrastructure and tools necessary to
integrate a 7900HT instrument into a laboratory workflow made up of existing data
management solutions.
The SDS Enterprise Database supports a basic two-tier, server-client architecture
within an Ethernet (10Base-T) or Fast Ethernet (100Base-T) network environment.
The database can function as part of an isolated star network via the SMC router
provided with the database (Figure 1-14), or as a node on a larger bus network
(Figure 1-15). The server computer uses a static IP address to simultaneously serve
up to 5 clients configured for either DHCP or static TCP/IP operation.
1-22
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About the SDS Enterprise Database Feature
LAN Architecture Example
In a basic configuration, the SDS Enterprise Database can serve up to four DHCP or
TCP/IP clients configured in a star topography. In this configuration, the server is
linked to the clients via the router provided with the database which has been
configured with a static IP address.
Note: The router features a WAN connection for connecting to an external bus.
Data Collection
Analysis
• Run Data
• Analysis Sessions
• Existing Plate Documents
• Template Documents
Opertaing System
Windows ® 2000
ABI
PRISM
7900HT
Applied
Biosystems
7900HT FastDetection
Real-Time
Sequence
PCR System instruments
System Instruments
SDS Enterprise
Database
Database Server
• Studies
• New Plate Documents
• Template Documents
Desktop Computers
Opertaing System
Windows ® 2000
Windows ® 2000
TCP/IP (Port 1521)
Network Protocol
TCP/IP
Client Software
SDS Client Software
or Automation
Controller Software
• Analysis Sessions
• Run Data
Network Protocol
TCP/IP
SDS Enterprise
Database Software
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Basic LAN Architecture for the SDS Enterprise Database
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1-23
Chapter 1 Product Overview
WAN Architecture Example
The SDS Enterprise Database can serve up to 5 clients as a node on a bus network.
Again, the server must be configured with a static IP address for identification on the
network and is linked to the DHCP or TCP/IP clients via the router provided with the
database.
Analysis
Data Collection
• Run Data
• Analysis Sessions
• Existing Plate Documents
• Template Documents
Opertaing System
Windows ® 2000
Network Protocol
TCP/IP
Applied
Biosystems
ABI PRISM
7900HT
7900HT Fast Real-Time
Sequence
PCR SystemDetection
instruments
System Instruments
• Analysis Sessions
• Run Data
SDS Enterprise
Database
Database Server
• Studies
• New Plate Documents
• Template Documents
Desktop Computers
Windows ® 2000
Network Protocol
TCP/IP
TCP/IP (Port 1521)
SDS Enterprise
Database Software
Client Software
SDS Client Software
or Automation
Controller Software
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0
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Office
WAN Architecture for the SDS Enterprise Database
DRAFT
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November 19, 2007 11:09 am, CH_Overview.fm
About the SDS Enterprise Database Software Suite
About the SDS Enterprise Database Software Suite
Database
Management
Software
The SDS Enterprise Database provides software utilities to help integrate the
database into laboratory workflows as describes in:
Database Modules for Large-Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25
Database Management Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26
Database Modules for Large-Scale Analysis
About the
Analysis Modules
Applied Biosystems offers two analysis modules (the SNP Manager Software and
RQ Manager Software) for conducting medium- to large-scale analysis of
SNP/genotyping and gene expression on the SDS Enterprise Database (Figure 1-16).
SNP Manager
Software
SNP/Genotyping
Study
Studies/Run Data
SDS Enterprise
Database
Analysis Sessions
RQ Manager
Software
Figure 1-16
Relative
Quantification Study
SDS Enterprise Database Applications for Large-Scale Analysis
About the
SNP Manager
Software
The SNP Manager Software is a stand-alone application used to perform medium- to
large-scale analyses of SNP (genotyping) data stored on the database. The software
can make comparative analyses of up to 5,000 plates or approximately 1.9 million
5´ nuclease reactions run on an Applied Biosystems 7900HT Fast Real-Time PCR
System. See the SNP Manager Software User Guide (PN 4351671) for more
information.
About the
RQ Manager
Software
The RQ Manager Software is a stand-alone application used to perform medium- to
large-scale analyses of relative quantification (gene expression) data stored on an
SDS Enterprise Database. The software can make comparative analyses of over
97 detectors (96 target genes and 1 endogenous control) across 200 plates, or
approximately 153,600 multiplexed reactions run on an Applied Biosystems 7900HT
Fast Real-Time PCR System. See the RQ Manager Software User Guide
(PN 4351670) for more information.
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1-25
Chapter 1 Product Overview
Database Management Utilities
Functional
Summary
Plate
Documents
SDS
Document
Creator
The SDS Enterprise Database includes a suite of software applications that
administer the use and content of the database. Figure 1-17 summarizes the basic
function of each tool and the type of data it handles.
SDS
Document
Loader
Plate
Documents
SDS
Template
Loader
SDS
Database
Archiver
Archive
Files (.arc)
Plate
Documents
Figure 1-17
SDS Document
Command Line
Tools
SDS User
Account
Manager
SDS Enterprise
Database
SDS
Document
Extractor
Plate
Document
Templates
Functional Summary of the Database Management Utilities
The SDS Enterprise Database Software Suite includes four command-line utilities
for managing plate documents, plate document templates, and run data produced by
the 7900HT instrument.
• SDS Document Creator – Creates plate documents in the database using the
contents of a plate document template and an assay plate setup file
• SDS Document Loader – Populates the database with the contents of existing plate
documents
• SDS Document Extractor – Creates plate documents from the data contained in the
database
• SDS Template Loader – Populates the database with the contents of existing plate
document templates
For detailed information on these utilities, see the SDS Enterprise Database for the
Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide
(PN 4351669).
SDS Database
Archiver
1-26
The SDS Archiver Software installs with the version of the SDS software that
includes the database client software. The software allows users with Administrator
privileges to download session (run) data from the database, create archive files from
them, and restore archived data to the database. See the SDS Enterprise Database for
the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide
(PN 4351669) for more information.
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November 19, 2007 11:09 am, CH_Overview.fm
Database Design and Information Management
SDS User
Account Manager
The SDS User Account Manager Software installs with the version of the SDS
software that includes the database client software. The software allows users of the
Administrator User Group to assign privileges and passwords for user accounts, and
to add and remove them. See the SDS Enterprise Database for the Applied
Biosystems 7900HT Fast Real-Time PCR System Administrators Guide
(PN 4351669) for more information.
Database Design and Information Management
About the
Study-Centric
Design
The SDS Enterprise Database persists three primary types of retrievable data used to
operate the 7900HT instrument and to analyze the collected run data:
• Plate documents
• Analysis sessions
• Studies
Figure 1-18 describes the sources and uses of the different types of data persisted to
the database throughout the operation of the Applied Biosystems 7900HT Fast
Real-Time PCR System.
1
Plate documents are
created on a client
computer using the SDS
software and saved to the
SDS Enterprise Database
Plate
Documents
Desktop
Computer
Database
Server
Plate
Documents
2
Prepared plates are sealed, and loaded
into the stacks of the Automation
Accessory. Just before each run, the
Automation Controller Software loads
the plate and scans its bar code. The
software then automatically downloads
the plate document with the matching
bar code from the database.
7900HT
Instrument
GR2179
7900HT raven
instrument front
F1
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7900HT FAST Real-Time PCR System
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5
The RQ Manager, SNP
Manager, or SDS software
is used to download and
analyze analysis sessions
or studies.
Database
Server
• Studies
• Run Data
Figure 1-18
• Run Data
• Analysis
Sessions
3
The software runs each plate
using the method stored in
the downloaded plate
document. During the run,
the software saves the raw
data to the plate document in
the database.
4
Immediately after each run, the software
performs a primary analysis
(multicomponenting and normalization)
of the raw data. The software then saves
the analyzed data to the database as an
analysis session and attaches it to the
appropriate study.
SDS Enterprise Database Data Flow Diagram
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November 19, 2007 11:09 am, CH_Overview.fm
1-27
Chapter 1 Product Overview
Types of
Retrievable Data
Persisted Plate Documents
Persisted plate documents include the equivalent information stored in SDS plate
documents. Plate documents have a 1:1 correspondence with the optical plate or Low
Density Array they represent. Each persisted plate documents consists of a collection
of plate setup information for running a plate on the 7900HT instrument including:
•
•
•
•
Sample/well assignments,
Marker/detector definitions and well assignments,
Marker/detector tasks and quantity,
Plate bar code
Analysis Sessions
An analysis session is a set of analysis results derived from a single raw data set
associated with a single plate document analyzed by the SDS software. Multiple
analysis sessions can be associated with the same plate document. Each analysis
session has a unique name used to distinguish it from the other sessions for the same
plate document.
Session data exists in two states on the database: attached or unattached. Attached
sessions are those that have been assigned to a specific study. Session data can be
attached to a study by the Automation Controller Software or by using the RQ
Manager or SNP Manager Software.
IMPORTANT! After you attach a session to a study, you cannot add it to another
study until you unattach it. See the RQ Manager Software User Guide or SNP
Manager Software User Guide for instructions on removing attached sessions from a
study.
RQ and SNP Studies
The SDS Enterprise Database software uses a system of studies to organize the
secondary analysis of session data produced by an Applied Biosystems 7900HT Fast
Real-Time PCR System. The central purpose of using the study system is to group
the analysis session data for analysis using the SNP Manager or the RQ Manager
Analysis Software.
Supporting API Documentation
Technology
Support
The SDS Enterprise Database includes a copy of the SDS Enterprise Database for
the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide,
which provides information for integrating the database into a laboratory workflow.
The guide includes:
• Detailed information about the SDS Enterprise Database API
• Supporting information such as sample code, javadoc reference, and
connectivity requirements for the database.
1-28
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November 19, 2007 11:09 am, CH_Overview.fm
Getting Started
In This Chapter
2
2
Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Using the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
Using SDS Plate Documents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29
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2-1
Chapter 2 Getting Started
Getting Started
Before You Begin
If this is your first time using the Applied Biosystems 7900HT Fast Real-Time PCR
System, consider completing the “Basic Software Skills Tutorial” on page 2-10
before continuing. The tutorial will provide you with the fundamental knowledge
required to operate the SDS software and will teach you timesaving techniques to
allow you to use it quickly and efficiently.
Procedure
Quick Reference
The following table contains a list of major procedures described inside this manual.
Setting Up and Running SDS Experiments
Creating an SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-8
Running an Individual SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23
Running Batches of SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-31
Stopping a Run from the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-28
Stopping a Run from the Automation Controller Software . . . . . . . . . . . . . . . . .4-41
Ejecting a Plate from the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-29
Ejecting a Plate from the Automation Controller Software . . . . . . . . . . . . . . . . .4-41
Analyzing Run Data
Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5
Analyzing an Absolute Quantification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5
Analyzing an Relative Quantification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15
Analyzing a Dissociation Curve (Melting Curve) Run . . . . . . . . . . . . . . . . . . . . .6-37
Maintaining the 7900HT Instrument
Changing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-12
Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-6
Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-14
Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-16
Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-20
Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-27
Verifying Instrument Performance Using a TaqMan® RNase P Instrument
Verification Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-30
Maintaining the Automation Accessory
Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . .7-37
Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-41
Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . .7-49
Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-52
Note: The Automation Accessory includes the Zymark® Twister Microplate Handler
and the fixed-position bar code reader. See “Instrument Components” on page 1-3
for more information.
2-2
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Getting Started
Using the SDS
Software
Online Help
The Sequence Detection Systems Software Online Help can guide you through the
procedures for setting up, performing, and analyzing runs. To get help at any time,
click
(Help button) located inside the dialog box or window in which you are
working.
The SDS software provides two ways to access the online help:
For More
Information
To…
Then…
access general help
select Help > SDS Online Help.
get help for using a specific dialog box,
plot, or feature
click
(help button) located inside the dialog
box or window in which you are working.
For information about the Applied Biosystems 7900HT Fast Real-Time PCR System
or the SDS software, Applied Biosystems recommends the following references:
• Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation
Guide (PN 4317595)
• Applied Biosystems 7900HT Fast Real-Time PCR System Quick Starts for:
– Allelic Discrimination (PN 4351673)
– Absolute Quantification (PN 4351672)
– Relative Quantification (PN 4351674)
• Sequence Detection Systems Software Online Help
• Microsoft Windows Operating System Online Help
• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time
PCR System Administrators Guide (PN 4351669)
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2-3
Chapter 2 Getting Started
Powering On the 7900HT Instrument
Powering On the
Instrument
The activation of the Applied Biosystems 7900HT Fast Real-Time PCR System is
sequential. You must activate each component in a specific order for the system to
initialize properly. If performed out of sequence, the components may not be able to
establish the necessary communication connections required for operation.
GR2009
7900HT
Front view with Robot
Status lights
Computer
power button
7900HT FAST Real-Time PCR System
7900HT instrument
power button
Monitor
power button
GR2009
Zymark Twister Microplate
Handler (power button in rear)
Figure 2-1 Power Buttons of the Applied Biosystems 7900HT Fast Real-Time
PCR System
IMPORTANT! Power on the power to the instrument and the Plate Handler at least
10 min before use. When activated, the instrument heats the sample block cover to
105 °C. If you start a run before the heated cover reaches 105 °C, the instrument will
pause until it reaches the optimal temperature before commencing the run.
1. Power on the monitor and computer.
2. Power on the Zymark Twister Microplate Handler by pressing the power switch
located on the back panel of the Plate Handler (see below).
PHYSICAL HAZARD. Keep clear of the arm when
activating the Plate Handler. Once activated, the arm automatically moves to its
home position.
Power switch
HI-POT
B
C
D
GR1728
A
Rear Panel of the Twister
If operating normally, the Plate Handler moves the arm to the home position
(over the output stack).
3. Power on the 7900HT instrument by pressing the power button located below
the status lights on the front of the instrument (see Figure 2-1).
If operating normally, the 7900HT instrument will do the following on startup:
• Emit a high-pitched tone signaling that the instrument initialized successfully.
2-4
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Powering On the 7900HT Instrument
• Cycle the status lights (Red, Orange, Green) indicating that the 7900HT
instrument is active (see “Reading the Instrument Status Lights” on page 2-5
for more information).
IMPORTANT! Do not power on the 7900HT instrument if you have removed the
lower-side panel of the instrument. Doing so prevents the instrument from uploading
the firmware from the computer and causes the software to display an error.
Reading the
Instrument
Status Lights
The 7900HT instrument contains three lights located on the lower-left side of the
front panel to indicate the status of the instrument (see Figure 2-2).
Status lights
6932RG
TH0097
Power button
Figure 2-2
Table 2-1
Status Lights of the 7900HT Instrument
Instrument Status Lights
Light/Appearance
Solid
Action
The 7900HT instrument is on
and in idle state (ready to run)
None
This state indicates normal
instrument function.
Flashing
• Interlocks are open and/or
the scan head has not
reached the safe position.
• The instrument door is open.
Flashing
The 7900HT instrument is
transmitting/receiving data
to/from the computer
(usually during a run).
None
If the light remains on during
startup for more than 2 min:
1. Check that the computer is
powered on and connected
to the instrument. (See
page 1-10 for a diagram of
instrument connections.)
(Green)
(Orange)
Status
Solid
This state indicates normal
instrument function.
• The instrument did not boot
properly, or
• 7900HT instrument has
experienced a system failure
2. If so, power off the
instrument, wait for 30 sec,
and then restart as explained
on page 2-4.
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Chapter 2 Getting Started
Table 2-1
Instrument Status Lights
Light/Appearance
Solid
Status
Action
The 7900HT instrument has
detected a fatal problem.
Power off the instrument, wait
for 30 sec, and then restart.
Note: If the problem persists,
(Red)
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Technical Support.
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
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Using the SDS Software
Using the SDS Software
Starting the
Software
1. Do one of the following:
• Select
> Programs > Applied Biosystems >
SDS 2.2.1.
• Double-click
(SDS 2.2.1) on the computer desktop.
SDS 2.2.1 >
If you are using an SDS Enterprise Database on your local area network, the
software will automatically prompt you to log in.
2. (Database Only) In the Login dialog box, enter your user name and password,
and click
.
Note: When working with a database, the SDS software requires that all users
have a user account and password (see the SDS Enterprise Database for the
Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide
(PN 4351669) for more information on assigning and modifying user accounts.)
The computer starts the SDS software and attempts to establish communication with
the 7900HT instrument. If the connection is successful, the software displays the
Connected icon (
) in the status bar when a plate document is open.
See “About the Message Bar” on page 2-9 for more information.
About the
Software
Interface
All software operations and displayed information occur inside the workspace of the
SDS software. The workspace provides quick access to all elements of the software
through the menubar and a pair of toolbars. Figure 2-3 summarizes the features of the
user interface of the SDS software.
Menubar
Contains a directory of menus
governing the operation of the
software
Window buttons
Minimize ( ), maximize (
or close ( ) the window
),
General Toolbar
Contains icons for controlling
the basic functions of the
software (see page 2-8)
Display Toolbar
Contains icons for controlling
the display of information in
the SDS software workspace
(see page 2-8)
Message Bar
Displays messages that indicate
the status of the instrument and
the SDS software (see page 2-9)
Instrument Connection Icon
Indicates the status of the
connection to the 7900HT
instrument (see page 2-9)
Figure 2-3
Elements of the SDS Software User Interface
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Chapter 2 Getting Started
About the
General Toolbar
The general toolbar contains tools used to control the basic functions of the software
for data management and analysis. The following list summarizes the functions of
the tools and provides a reference in parenthesis ( ) to a procedure involving the
function.
– Creates a new plate document (see page 2-13)
– Opens a plate document from the hard drive (see page 2-19)
– Saves the plate document to the hard drive (see page 2-17)
– Opens a plate document from the database (see page 2-19)
– Saves the plate document to the database (see page 2-17)
IMPORTANT! The general toolbar will display the
and
options only if
the SDS software is connected to an SDS Enterprise Database.
– Imports data from a text file (see page 2-16)
– Exports data to a text file (see page 2-14)
– Opens the Print Report dialog box (see page 2-13)
– Opens the Find tool (see page 2-13)
– Removes the selected object and places it into memory (see page 2-13)
– Copies the selected object into memory (see page 2-13)
– Inserts the object in memory into the current selection (see page 2-13)
– Analyzes the plate document (see page 2-13)
– Opens the Analysis Settings dialog box (see page 2-13)
About the
Display Toolbar
The display toolbar contains tools used to control the display of information inside
the SDS software workspace. The following list summarizes the functions of the
tools and provides a reference in parenthesis ( ) to a procedure involving the function.
– Hides/shows the Well Inspector Panel (see page 2-12)
– Hides/shows the Plate List Panel (see page 2-12)
– Hides/shows the Plate Grid (see page 2-12)
– Hides/shows the Table Pane (see page 2-12)
– Hides/shows the System Raw Data Pane (see page 2-12)
– Hides/shows the Multicomponent Plot (see page 2-12)
– Hides/shows the Amplification Plot (see page 2-12)
– Hides/shows the Standard Curve Plot (see page 2-12)
– Hides/shows the Gene Expression Plot (see page 2-12)
– Hides/shows the Dissociation Plot (see page 2-12)
– Zooms the plate grid (see page 2-12)
– Opens the Display Settings dialog box (see page 2-12)
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Using the SDS Software
About the
Message Bar
The message bar displays a variety of messages to indicate the status of the
instrument and software. The following table summarizes all of the messages
displayed in the Message bar.
Table 2-2
Status Messages
Message
Then the SDS software is…
Ready
ready and awaiting instructions.
Collecting Data
currently running a plate document.
Reanalyze data
requesting analysis of plate document data.
The analysis settings for the plate document have
been changed and the document requires reanalysis
for them to take effect.
Analyzing data... + Progress bar
completing the calculations for the current analysis.
The metered bar to the right of the message displays
the progress of the analysis.
Saving data... + Progress bar
saving the plate document or plate document
template to a storage device.
The metered bar to the right of the message displays
the progress of the action.
Importing data...
importing a file.
The metered bar to the right of the message displays
the progress of the action.
Exporting data... + Progress bar
exporting the data inside the current plate document
to a file.
The metered bar to the right of the message displays
the progress of the action.
About the
Instrument
Status Icon
The Instrument Status Icon indicates the status of the connection to the 7900HT
instrument.
– The computer has successfully connected to the instrument
and is awaiting a command
– The computer is not able to connect to the instrument (the
instrument is not connected or is powered off)
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Chapter 2 Getting Started
Basic Software Skills Tutorial
User Access
Requirement
About This
Tutorial
Accessing the
Online Tutorial
If using a computer linked to an SDS Enterprise Database, you must have Scientist or
Administrator access privileges to complete this tutorial.
This tutorial will:
•
•
•
•
•
Teach you to create, save, print, export, and import SDS plate documents
Familiarize you with the basic components of the SDS software interface
Explain how to customize and arrange the user interface to suit your needs
Teach you to use the hand-held bar code reader
Provide you with timesaving techniques that will increase your effectiveness
with the SDS software
The Sequence Detection Systems Software Online Help provides a version of this
tutorial. If you prefer to follow the online tutorial, open the online help:
1. If not already active, start the SDS software as explained on page 2-7.
2. Select Help > SDS Online Help.
3. In the SDS Online Help, click Basic Skills Tutorial from the list of options.
4. Follow the directions displayed on your screen.
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Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when performing
the lessons in this tutorial. This section describes the types of actions you may need
to perform depending on how your administrator has configured the database.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
If the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 2-4
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
If the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 2-5
Electronic Signature Verification Dialog Box Options
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Chapter 2 Getting Started
Lesson 1: Using Plate Documents
About SDS Plate
Documents
Every plate run on the Applied Biosystems 7900HT Fast Real-Time PCR System
requires the creation of a plate document using the SDS software. A plate document
is a virtual representation of a consumable used to contain samples and reagents
during a sequence detection run. The software uses the plate document to coordinate
the operation of the instrument (thermal cycling and data collection), to organize and
store the data gathered during the PCR, and to analyze the run data.
Table 2-3
Plate Document Types of the SDS Software
File
Ext.
Icon
Description
SDS 7900HT
Document
*.sds
Plate Documents are the primary file you will use.
They are generated for every kind of experiment
and are generally used to run plates.
SDS 7900HT
Template
Document
*.sdt
Although optional, plate document templates are
useful as timesaving devices for experiments where
samples are run on plates with identical assay
configurations.
SDS 7900HT
Multiple Plate
Document
*.sdm
Multiple plate documents are used to analyze data
from relative quantification experiments. The use of
multiple plate documents is discussed in detail in
Chapter 6, “Analyzing Real-Time Data.”
The exercises in this lesson will familiarize you with the use of plate documents.
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Exercise 1:
Creating a Plate
Document
You will need to create a plate document for every plate you run on the 7900HT
instrument.
IMPORTANT! The SDS software can handle multiple documents simultaneously,
however the processing speed of your computer will decrease with each open
document. For that reason, Applied Biosystems recommends limiting the number of
open documents to no more than 10.
1. If not already active, start the SDS software:
a. Select
>
SDS 2.2.1).
Programs >
Applied Biosystems >
SDS 2.2.1 >
b. (Database Only) In the Login dialog box, enter your user name and
password, and click
.
2. Click
(or select File > New).
3. Configure the New Document dialog box with the following settings:
• Assay drop-down list – Select Absolute Quantification.
• Container drop-down list – Select 96 Wells Clear Plate.
• Template drop-down list – Select Blank Template.
• Barcode field – Enter Practice.
Note: Normally, you would enter a file name or bar code consisting of up to
128-characters in the Barcode field, However, for the purpose of demonstrating
the software function, this tutorial instructs you to enter “Practice.”
4. Click
.
The software displays a new plate document with appropriate attributes.
5. Open the plate document template for the TaqMan® RNase P Instrument
Verification Plate (PN 4310982, for the Standard 96-Well Block) and export its
plate setup as explained on page 2-14.
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Chapter 2 Getting Started
Exercise 2:
Exporting Data
from a Plate
Document
As an alternative to setting up plate documents manually (as described in Chapter 4,
Operating the Instrument), the SDS software can import plate document setup
information directly from tab-delimited Assay Plate Setup Files. You can export
Assay Plate Setup Files from the SDS software or create them using a third-party
application such as spreadsheet or LIMS software (see Appendix A, “Software
Reference,” for more information). In the following exercise, you will export the
Assay Plate Setup File for a plate document template so that you will import in
“Exercise 3: Importing Setup Table Data into a Plate Document” on page 2-16.
Note: In addition to Assay Plate Setup Files, the SDS software can also export data
from most of the analysis plots, graphs, and tables. See Appendix A, “Software
Reference,” for more information.
Opening the Plate Document Template
In the following procedure, you will open the plate document template for a
TaqMan® RNase P Instrument Verification Plate. The SDS software automatically
installs several default plate document templates to:
\AppliedBiosystems\SDS2.2.1\Templates. In addition to the RNase P Plate document
template, Applied Biosystems provides in the same directory several plate document
templates for plates run regularly on the 7900HT instrument.
1. Click
(or select File > Open).
2. In the Look In field of the Open dialog box, navigate to:
\AppliedBiosystems\SDS2.2.1\Templates.
3. Select File of type > SDS 7900HT Template Document (*.sdt).
4. In the Look In field, select the 96 Well RNaseP Install Plate.sdt file.
Select
5. Click
.
The software opens the plate document template.
6. Export the plate setup information from the plate document template as
explained on page 2-15.
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Exporting the Plate Setup Information
1. Click
(or select File > Export).
2. In the Look In field of the Export dialog box, navigate to:
Applied Biosystems/SDS Documents.
3. In the Export dialog box, select Export > Setup Table.
4. Select the All Wells radio button.
5. Select the SDS 2.2.1 radio button.
6. In the File name field, enter Practice.
7. Click
.
The software saves the plate document setup table information to a
tab-delimited text file entitled ‘Practice.txt’.
Note: If desired, you can open the ‘Practice.txt’ Assay Plate Setup File using
the Microsoft Notepad, Wordpad, or Word software to view its contents.
8. Select File > Close to close the plate document template.
IMPORTANT! Do not close the plate document created in “Exercise 1: Creating
a Plate Document” on page 2-13.
9. Import the exported plate setup information into the plate document as
explained on page 2-16.
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Chapter 2 Getting Started
Exercise 3:
Importing Setup
Table Data into a
Plate Document
As a timesaving device, the SDS software allows you to import plate document setup
information from exported or fabricated Assay Plate Setup Files. To illustrate this
feature, import the plate grid setup information contained in the Practice.txt file
(from “Exercise 2: Exporting Data from a Plate Document” on page 2-14) into the
empty plate document created in “Exercise 1: Creating a Plate Document” on
page 2-13.
1. If the plate document from Exercise 1 is still open, go to step 2. Otherwise,
create a new plate document to receive the setup table data:
a. Click
(or select File > New).
b. Configure the New Document dialog box with the same settings as the
plate document template:
Assay drop-down list – Select Absolute Quantification.
Container drop-down list – Select 96 Wells Clear Plate.
Template drop-down list – Select Blank Template.
Barcode field – Leave blank.
c. Click
.
The software displays a new plate document with appropriate attributes.
2. Click
(or select File > Import).
3. In the Look In field of the Import dialog box, navigate to:
Applied Biosystems/SDS Documents.
4. Select the Practice.txt file created in Exercise 5.
5. Click
.
The software imports the setup information of the Practice.txt file into the plate
grid and table of the empty plate document.
Note: For more information on importing and exporting setup table data using
the SDS software, see Appendix A, “Software Reference.”
6. Save the plate document as explained on page 2-17.
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Exercise 4:
Saving a Plate
Document
The Save command stores any changes to the plate document setup information and
display settings. If using the version of the SDS software that includes the client
software for the SDS Enterprise Database, you also have the option of saving the
plate document file to the database.
Choose from the procedures below:
• If using a database, save the plate document to the database as described on
page 2-18.
• If you are not using a database, save the plate document to your computer hard
drive as described below.
Saving the Plate Document as an SDS 7900HT Document
1. Click
(or select File > Save As).
2. Select File of type > SDS 7900HT Document (*.sds).
3. In the File name field, enter Practice.
4. Click
.
5. In the Document not properly set up warning, click
.
The software saves the plate document to a file entitled Practice.sds.
6. Close the saved plate document file as explained on page 2-18.
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Chapter 2 Getting Started
Saving the Plate Document to the SDS Enterprise Database
1. Click
(or select File > Save Document to Database).
2. In the Document not properly set up warning, click
.
3. In the Save Document to database dialog box, leave the Comment field blank or
enter a brief comment describing the file.
Note: The Comment field can contain up to a 256-character description of the
file.
4. Click
to save the file.
5. In the Document not properly set up warning, click
6. In the Saved Document dialog box, click
.
.
7. Select File > Close.
8. If prompted to save the plate document, click
.
The SDS software closes the file without saving it.
9. Open a plate document file as explained on page 2-19.
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Exercise 5:
Opening a Plate
Document
In this exercise, you will be opening two documents that you will use in the following
exercises: the plate document (from Exercise 3), and a plate document template. The
purpose of this lesson is to familiarize you with the tasks of retrieving plate document
that have been saved to the computer hard drive or the SDS Enterprise Database.
Choose from the procedures below:
• If using a database, save the plate document to the database as described on
page 2-18.
• If you are not using a database, save the plate document to your computer hard
drive as described below.
Opening the SDS Plate Document File
1. Click
(or select File > Open).
2. In the Look In field of the Open dialog box, navigate to:
Applied Biosystems/SDS Documents
3. In the Look In field, select the Practice.sds file.
Select
4. Click
.
The software opens the Practice.sds file.
5. Select File > Close.
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Chapter 2 Getting Started
Opening the Plate Document from the SDS Enterprise Database
1. Click
(or select File > Open Document from Database).
2. Configure the Find Plates Matching These Criteria table of the Plate Query
dialog box:
a. Click the first cell in the Variable column, and select Barcode.
b. Click the first cell in the Conditions column, and select Contains.
c. Click the first cell in the Value1 column, and enter Practice.
3. Click
.
Select
Barcode
Select
Contains
Enter
Practice
Click to begin the search
4. In the Search Results list, select the Practice plate document, and click
Click to select
the document
Click to open the document
5. Click
.
6. Go on to “Lesson 2: Viewing and Resizing Panes” on page 2-21.
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Lesson 2: Viewing and Resizing Panes
Overview
Exercise 1:
Resizing Panes,
Views, and Plots
Because plate documents can display setup and analysis data in multiple views
simultaneously, the SDS software provides several navigational devices to help
manage the information. This lesson will teach you to use the different aids to reduce
screen clutter and ensure efficient use of the software.
You can resize the panes, views, and plots of plate documents by moving the grey
lines dividing them horizontally and vertically.
Dividing line
(click and drag)
Dividing line (click and drag)
1. Click and drag the grey line dividing the plate grid and well inspector to the right.
The software expands the plate grid and table pane to the new width.
2. Using the grey dividing lines, resize other elements of the plate document until
you are comfortable using the feature.
Exercise 2: Hiding
and Showing
Panes, Views,
and Plots
You can also toggle the presence of the plate document panes, views, and plots using
the icons in the Display toolbar.
1. In the Display toolbar, click
(the Hide/Show Table Pane button).
The software removes the table pane from the plate document.
2. Click
again to show the table pane.
The software restores the table pane to the plate document.
The display toolbar can be particularly useful for manipulating information
shown in the plate document. See “About the Display Toolbar” on page 2-8 for a
list of the other icons of the display toolbar and the panes they control.
3. Practice hiding and showing the other plate document elements by clicking
other buttons in the Display toolbar until you are comfortable using the feature.
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Chapter 2 Getting Started
Exercise 3:
Maximizing/
Minimizing
Panes, Views,
and Plots
You can maximize the panes, views, and plots of plate documents by clicking the
sizing buttons embedded inside the grey dividing lines.
Note: Sizing buttons are the arrowhead marks (
) that appear between adjacent
elements of the plate document. When clicked, a sizing button hides the element to
which it points.
1. Click (down-arrow sizing button) in the divider between the plate grid and the
table pane to maximize the plate grid vertically.
The software maximizes the plate grid by minimizing the table pane.
Sizing button
2. Click and drag the grey divider at the bottom of the plate document to restore the
table pane to its original size (using the action described in Exercise 1).
3. Using the sizing buttons, maximize/minimize other elements of the plate
document until you are comfortable using the feature.
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Lesson 3: Using the Plate Grid
Overview
The plate grid (see Figure 2-6) is an important interface tool for the SDS software.
The software uses the grid to convey information about the plate and allows you to
select specific wells for viewing and analysis. The following exercises will teach you
how to use and modify the elements of the plate grid.
Plate grid
Figure 2-6
Exercise 1:
Viewing Well
Information
Plate Grid of SDS Plate Documents
The SDS software provides two methods for viewing the information associated with
a well or wells of the plate document. To view the information for a well of the plate
document, do one of the following:
• Click any well in the plate grid to select it.
When selected, the software outlines the well in black and displays the
associated information in the well inspector of the Setup tab.
Setup tab displays the
information of the selected well
• Move the pointer over any well of the plate grid.
The software displays the information for the well in a yellow pop-up window.
Pop-up displays the information
of the well to which the mouse
cursor points
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Chapter 2 Getting Started
Exercise 2:
Selecting Multiple
Wells
The SDS software provides several methods for selecting wells from the plate grid.
The following exercise will familiarize you with most of them.
1. Select a block of wells from the plate grid by doing one of the following:
• Click and drag the mouse cursor across the block of wells, or
• Click the well at the top-left corner of the block. Then while holding-down
the Shift key, click the well at the bottom-right corner of the block.
The software outlines the selected wells with a black border.
2. Select several isolated wells of the plate grid by doing of the following:
a. Hold-down the Ctrl key, and click individual wells to select them.
The software outlines the selected wells with a black border.
b. While holding down the Ctrl key, click the wells to de-select them.
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3. Select an entire column or row of wells using the headers:
a. Click the header for row B to select all wells in the row.
The software outlines the wells of row B with a black border.
b. Press and hold either the Shift or Ctrl key, then click other columns or row
headers to select multiple columns.
Row B header
or
4. Select all wells of the plate grid by clicking the top-left corner of the plate grid.
The software outlines all of the wells in the plate document with a black border.
Button
5. Using the techniques illustrated in steps 1 to 4, practice selecting portions of the
plate grid until you are comfortable using the feature.
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Chapter 2 Getting Started
Exercise 3:
Zooming the
Plate Grid
You can zoom the plate grid to display the well information by clicking the Zoom
Grid button ( ).
1. Click
and observe how the grid expands to display the well information.
2. Click
again to restore the plate grid to the original size.
3. Practice zooming portions of the plate grid until you are comfortable using the
feature.
Exercise 4:
Resizing Wells
Using the Border
Lines
You can also adjust the size of the plate grid wells by moving the lines in the row or
column headers.
1. Move the mouse cursor over a border line in the row or column header.
The mouse cursor becomes a double arrow (
).
2. Click and drag the mouse cursor to adjust the well to a new width.
The software resizes all wells in the plate grid to match the new width.
Mouse cursor
3. Practice resizing the wells of the plate grid until you are comfortable using the
feature.
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Lesson 4: Using the Hand-Held Bar Code Reader
Overview
The hand-held bar code reader functions as an extension of the keyboard that you can
use to automatically enter bar codes into the SDS software. When the reader is used
successfully to scan a bar code, it automatically:
• Transmits the alphanumeric equivalent of the bar code to the software. The
software enters the bar code text wherever the cursor is active.
• Transmits a carriage-return (the equivalent of pressing the Enter key).
Exercise:
Entering Bar
Code Information
Using the
Hand-Held Bar
Code Reader
The following procedure explains how to enter a bar code number using the had-held
bar code scanner. Normally, you would scan the bar code into the New Document
dialog box during plate document creation.
1. Select Tools > Document Information.
2. In the Document Information dialog box, click the Comments field.
Click here
3. While holding the hand-held bar code reader 20 to 30 cm away from a plate, aim
at the center of the bar code and press the trigger. The scanner emits a sweeping
laser beam that appears as a red line on the plate. Slowly move the scanning
beam slowly across the bar code until the scan gun emits a high-pitched tone
acknowledging that it has read the code.
LASER HAZARD. Exposure to direct or reflected laser
light can burn the retina and leave permanent blind spots. Never look into the
laser beam. Remove jewelry and anything else that can reflect the beam into
your eyes. Protect others from exposure to the beam.
PECY001DL3
GR2110
20-30 cm
After the gun has read the bar code, the software automatically populates the
selected field with the alphanumeric equivalent of the bar code.
4. Click
to close the Document Information dialog box.
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Chapter 2 Getting Started
Lesson 5: Using Contextual Menus
Overview
The SDS software features contextual menus as a timesaving device to provide
access to the commands for an associated view or pane.
To access a contextual menu, move the pointer over a pane or view of interest, then
right-click. The menu appears at the location of the pointer.
Contextual menu
Figure 2-7
Contextual Menu of the SDS Software
All contextual menus provide the following common commands:
Table 2-4
Standard Commands of the Contextual Menu
Command
Result
See Page
Hide <pane or plot>
Hides the pane or view.
2-21
Save <pane or plot>
to Image File
Opens the Export Graphic dialog box for exporting the
selected view or pane as a JPEG graphic file.
A-16
Display Settings
Opens the Display Settings dialog box, which allows
you to modify the appearance of the view, pane, or plot.
3-22
Lesson 6: Using Keyboard Shortcuts
Exercise: Closing
the Plate
Document
The SDS software provides keyboard shortcuts for invoking the major functions of the
software. A keyboard shortcut is a combination of two keys (Ctrl and another key)
that, when pressed in unison, instruct the software to perform an action. The software
lists the keyboard shortcuts next to the options in each menu on the menu bar.
To demonstrate the use of keyboard shortcuts, close the open plate document as follows:
1. Simultaneously press the Ctrl and W keys (Ctrl+W).
2. When prompted to save the plate document, click
.
Note: The Sequence Detection Systems Software Online Help contains a complete
list of the keyboard shortcuts for the SDS software. To view the list, open the online
help as explained in “Using the SDS Software Online Help” on page 2-3.
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Using SDS Plate Documents
Using SDS Plate Documents
Using Multiple
Plate Documents
Elements of a
Plate Document
Although the SDS software can handle multiple documents simultaneously, the
processing speed of the computer decreases with each open document. Consequently,
Applied Biosystems recommends limiting the number of open documents to 10.
Figure 2-8 shows a typical plate document and identifies its components which are
described on the following pages.
Plate grid
(see below)
Tabs
(page 2-30)
Well inspector
(page 2-31)
Table pane
(page 2-29)
Figure 2-8
Plate Grid
Common Elements of SDS Plate Documents
The cells of the plate grid correspond to the wells of the reaction device that the plate
document models. The grid displays well information according to the type of plate
document. The Plate Grid properties settings in the Display Settings dialog box
determine content and appearance of the information displayed inside the cells of the
grid.
Note: For more information on configuring the display settings for the plate grid,
click
Table Pane
(help button).
The table pane displays the setup and analysis properties for the plate document. You
can export the table pane as a tab-delimited text file for use by a spreadsheet
application (see Appendix A, “Software Reference,” for more information).
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Chapter 2 Getting Started
Tabs
Plate documents can have up to six tabs (depending on their function) as described
below:
Table 2-5
Tabs of the SDS Software
Tab
Function
Views/Plots
Setup
Displays well information, and allows you
to configure the plate grid with setup
information.
Well Inspector
Instrument
Used to program the plate document
method, run the plate document, or send it
to the Plate Queue.
• Method Editor
– Thermal Profile tab
– Auto Increment tab
– Ramp Rate tab
– Data Collection tab
• Real-Time tab
• Plate-Read tab
Raw Data
Plot
Displays the raw fluorescence collected
from the sequence detection run.
Raw Data Plot
The Raw Data tab is visible only in plate
documents containing run data.
To view the tab, open a plate document
containing run data, click
, then select
the Raw Data Plot tab.
Calibration
Data
Displays the Background and Pure Spectra
calibration data used for the signal
normalization and multicomponenting
analysis of the run data.
Calibration Data
• Background Plot
• Pure Dyes Plot
The Calibration Data tab is visible only in
plate documents containing run data.
To view the tab, open a plate document
containing run data, click
, then select
the Calibration Data tab.
Results
Displays analyzed run data.
Allelic Discrimination Data
The Analysis tab is visible only in plate
documents containing analyzed run data.
• Allelic Discrimination Plot
Absolute Quantification
• Amplification Plot
• Standard (Curve) Plot
Relative Quantification
• Amplification Plot
Dissociation
Curve
Displays analyzed dissociation curve data
from a programmed ramp.
Dissociation Plot
The tab is visible only in plate documents
containing analyzed data from a real-time
run with a programmed ramp.
2-30
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Using SDS Plate Documents
Well Inspector
The Well Inspector (Figure 2-9) applies detector and sample information to the wells
inside the grid pane and displays information from the selected cells in the plate grid.
Sample name field
Detector list
Passive Reference drop-down list
Omit Well check box
Figure 2-9
Components of the Well Inspector
• Sample Name text field – An editable field that displays the sample name
applied to the selected well(s)
Note: The Sample Name field will display *Mixed* if multiple wells with
different sample names are selected.
• Detector list – Lists all available detectors copied to the plate document
• Omit Well check box – Toggles the activity of the well. If selected, the software
eliminates the data from the selected well from all analysis procedures.
• Passive Reference drop-down list – Displays the fluorescent dye used as a
passive reference
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Chapter 2 Getting Started
2-32
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Preparing a Run
In This Chapter
3
3
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Quick Review: Powering On the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . . . 3-7
Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
Step 2 – Applying Detectors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Step 3 – Configuring the Plate Document with Tasks . . . . . . . . . . . . . . . . . . . . . 3-14
Step 4 – Setting the Passive Reference and Omitting Wells . . . . . . . . . . . . . . . . . 3-16
Step 5 – Programming the Plate Document Method . . . . . . . . . . . . . . . . . . . . . . . 3-17
Step 6 – Saving the Plate Document as a Template. . . . . . . . . . . . . . . . . . . . . . . . 3-22
Step 7 – Creating a Plate Document from the Template . . . . . . . . . . . . . . . . . . . . 3-24
Step 8 – Applying Sample and Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . 3-25
Step 9 – Running the Plate on the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . 3-26
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3-1
Chapter 3 Preparing a Run
Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when creating and
modifying plate documents as described in this chapter. This section describes the
types of actions you may need to perform depending on how your administrator has
configured the database. For more information on any of the features described
below, see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast
Real-Time PCR System Administrators Guide.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
When the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 3-1
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
When the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 3-2
3-2
Electronic Signature Verification Dialog Box Options
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Before You Begin
Before You Begin
Background
Information
Chapter 5, “Analyzing End-Point Data,” and Chapter 6, “Analyzing Real-Time Data,”
include brief discussions of the experiments that you can perform using the 7900HT
instrument. Before beginning, you may want to review the appropriate chapter for
your experiment:
Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5
Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5
Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15
Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37
Getting More
Information from
the SDS
Online Help
The Sequence Detection Systems Software Online Help can guide you through the
procedures for setting up, performing, and analyzing runs. To get help at any time,
click the
button located inside the dialog box or window in which you are
working.
Maximizing
Throughput for
End-Point Runs
For end-point applications such as allelic discrimination, the throughput of the
Applied Biosystems 7900HT Fast Real-Time PCR System can be increased by
dividing the workload between the 7900HT instrument and several thermal cyclers.
Unlike real-time runs, the 7900HT instrument collects data for end-point runs after
the completion of the PCR. Consequently, you can perform the thermal cycling of
end-point plates elsewhere and then transfer them to the 7900HT instrument
afterwards for data collection and analysis.
IMPORTANT! To perform the thermal cycling and the plate read using the 7900HT
instrument, run the plate first as a real-time plate document and then again as an
allelic discrimination plate document (see “Step 5 – Programming the Plate
Document Method” on page 3-17 for the procedure).
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Chapter 3 Preparing a Run
Workflow Overview
Experiments/
Runs Performed
on the 7900HT
Instrument
Absolute
Quantification
Workflow
Absolute Quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-16
Dissociation Curve (Melting Curve) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5
Pure Dye (Spectral Calibration). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-20
Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5
RNase P Instrument Performance Verification . . . . . . . . . . . . . . . . . . . . . . . . . . .7-30
1. Create an absolute quantification plate document (see page 3-8).
2. *Apply detectors to the plate document:
a. Create detectors for the absolute quantification probes (see page 3-9).
b. Copy the detectors to the plate document (see page 3-11).
3. Configure the plate document with tasks and quantities:
a. Configure the plate document with detector tasks (NTC, Standard, and
Unknown) (see page 3-14).
b. Assign quantities to the wells of the plate document that contain standards
(see page 3-15).
4. Program the method for the absolute quantification run (see page 3-17).
5. If performing an assay in which you would like to collect dissociation data, add a
temperature ramp to the thermal profile to perform a dissociation curve analysis
(see page 3-21).
6. Choose from the following:
– If running a single plate, then continue to step 2.
– If running the first plate in a series of plates with identical assay
configurations, then save the plate document as a template (see page 3-22).
7. Create a plate document from the template created in step 2 (see page 3-24).
8. Configure the document with sample names and plate information (see
page 3-25).
9. Prepare and run the absolute quantification plate or plates (see page 4-1).
10.Analyze the run data (see page 6-5).
*Steps 2 and 2 can be eliminated by importing the plate document setup information
from a tab-delimited text file. See “Importing Plate Document Setup Table Files”
on page A-2 for more information.
3-4
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Workflow Overview
Relative
Quantification
Workflow
1. Create a relative quantification plate document (see page 3-8).
2. *Apply detectors to the plate document:
a. Create detectors for the relative quantification probes (see page 3-9).
b. Copy the detectors to the plate document (see page 3-11).
3. *Configure the plate document with detector tasks (NTC and Unknown)
(see page 3-14).
4. *Program the method for the relative quantification run (see page 3-17).
5. Choose from the following:
– If running a single plate, then continue to step 2.
– If running the first plate in a series of plates with identical assay
configurations, then save the plate document as a template (see page 3-22).
6. Create a plate document from the template created in step 2 (see page 3-24).
7. Configure the document with sample names and plate information (see
page 3-25).
8. Prepare and run the relative quantification plate or plates (see page 4-1).
9. Analyze the run data (see page 6-15).
*Steps 2 and 2 can be eliminated by importing the plate document setup information
from a tab-delimited text file. See “Importing Plate Document Setup Table Files”
on page A-2 for more information.
Dissociation
(Melting) Curve
Workflow
The SDS software can perform a dissociation curve analysis as part of an absolute
quantification run. Therefore, to perform a dissociation curve, construct a plate
document for absolute quantification as explained on page 3-4 and configure the
method with a temperature ramp as explained on page 3-21.
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3-5
Chapter 3 Preparing a Run
Allelic
Discrimination
Workflow
1. Create an allelic discrimination plate document (see page 3-8).
2. *Apply markers to the plate document:
a. Create detectors for the allelic discrimination probes (see page 3-9).
b. Create a marker for each allelic discrimination probe pairing (see page 3-12).
c. Copy the marker(s) to the plate document (see page 3-13).
3. *Assign detector tasks to the wells of the plate document (NTC and Unknown)
(see page 3-14).
4. If you would like to perform thermal cycling of the allelic discrimination plate on
the 7900HT instrument, create a real-time plate document for the plate and
program it with the method for the allelic discrimination run. Otherwise, continue
to step 2 (see page 3-17).
5. Choose from the following:
– If running a single plate, then continue to step 2.
– If running the first plate in a series of plates with identical assay
configurations, then save the plate document as a template (see page 3-22).
6. Create a plate document from the template created in step 2 (see page 3-24).
7. Configure the document with sample names and plate information
(see page 3-25).
a. Prepare the allelic discrimination plate or plates and perform thermal cycling
on a designated thermal cycler (see page 4-1).
b. Run the allelic discrimination plate or plates on the 7900HT instrument.
8. Analyze the run data (see page 5-5).
*Steps 2 and 2 can be eliminated by importing the plate document setup information
from a tab-delimited text file. See “Importing Plate Document Setup Table Files”
on page A-2 for more information.
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Quick Review: Powering On the 7900HT Instrument
Quick Review: Powering On the 7900HT Instrument
Powering On the
Instrument
Note: The following table includes a set of abridged procedures for activating the
components of the Applied Biosystems 7900HT Fast Real-Time PCR System. For a
complete explanation of the procedure, see “Powering On the 7900HT Instrument”
on page 2-4.
IMPORTANT! Do not power on the 7900HT instrument while the lower-side panel of
the instrument is removed. Doing so prevents the instrument from uploading the
firmware from the computer and causes the software to display an error.
1. Power on the monitor and computer.
2. If using an automation module, power on the Zymark® Twister Microplate
Handler.
Power
HI-POT
B
C
D
GR1728
A
(Rear panel of the Twister)
3. Power on the 7900HT instrument.
6932RG
TH0097
Power button
4. Start the SDS software (select
> Programs >
Applied Biosystems > SDS 2.2.1 > SDS 2.2.1)
5. If using the SDS Enterprise Database, enter your user name and password in the
appropriate fields of the Login dialog box, then click
.
The SDS software displays the program window.
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3-7
Chapter 3 Preparing a Run
Step 1 – Creating a Plate Document
User Access
Requirement
About ABI PRISM
Plate Documents
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to create plate documents.
Every plate run on the 7900HT instrument requires the creation of a plate document
within the SDS software. Each plate document is a virtual representation of a
specific consumable (that is, a reaction plate or Low Density Array) containing
samples and reagents for use on the 7900HT instrument.
Plate documents contain the following information:
•
•
•
•
Creating a Plate
Document
Detector information and arrangement on the plate
Marker information and arrangement on the plate (allelic discrimination only)
Sample information and arrangement on the plate
Method parameters for the run (absolute and relative quantification)
1. If not already open, start the SDS software as explained on page 3-7.
2. Click
(or select File > New).
3. Configure the New Document dialog box with settings for the run:
• Assay drop-down list – Select the type of assay appropriate for your plate.
• Container drop-down list – Select the type of consumable you intend to run.
• Template drop-down list – Select Blank Template.
• Barcode field – Do one of the following:
If you are creating a:
– Plate document to run a single plate, scan or enter the bar code for the plate.
– Plate Document Template for creating multiple plates, leave the field
blank.
IMPORTANT! The SDS Enterprise Database does not support the creation of
two plate documents of different run types with the same bar code.
Note: If performing a dissociation curve experiment, select Absolute
Quantification from the Assay drop-down list.
Assay drop-down list
Container drop-down list
Template drop-down list
Barcode field
4. Click
.
5. Create and copy detectors (and markers) to the new plate document as described
in “Step 2 – Applying Detectors and Markers” on page 3-9.
3-8
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Step 2 – Applying Detectors and Markers
Step 2 – Applying Detectors and Markers
User Access
Requirement
Creating
Detectors
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to create and apply detectors and markers.
Before you can use a plate document to run a plate, it must be configured with
detector information for the experiment (and marker information if performing
allelic discrimination). A detector is a virtual representation of: a TaqMan® probe and
primer set used for detection of a single target nucleic acid sequence, or a primer set
utilizing SYBR® Green Double-Stranded DNA Binding Dye 1 (used for the detection
of double-stranded DNA product). Before using the plate document, you must create
and apply detectors for all assays present on the plate.
1. Select Tools > Detector Manager.
2. In the Detector Manager dialog box, click
.
3. Configure the Add Detector dialog box:
• Name field – Enter a name for the detector.
The name of the detector must be unique and should reflect the target locus
of the assay (such as GAPDH or RNase P). Do not use a name for more
than one detector. The SDS software does not distinguish between
detectors of the same name, even if they use a different dye set.
• Group field – (Optional) Enter or select a detector group for the detector.
• Description field – (Optional) Enter a brief description of the assay
(up to 32 characters).
• Reporter/Quencher drop-down lists – Select the appropriate reporter and
quencher dyes for the probe.
If creating a detector for an assay using the SYBR Green 1 dsDNA Binding
Dye, set the Quencher Dye drop down list to Non Fluorescent.
If you are using a custom dye not manufactured by Applied Biosystems,
you must create and run a pure dye plate for the dye before applying it to a
detector (see “Adding Custom Dyes to the Pure Dye Set” on page 7-27).
• Color box – (Optional) Click the box, then use the Color Picker dialog box
to select a color to represent the detector, and click
.
• Notes field – (Optional) Enter any additional comments for the detector
(up to 200 characters).
Name field
Group field
Description field
Reporter/Quencher drop-down lists
Color box
Notes field
Creation and Modification
date stamps
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3-9
Chapter 3 Preparing a Run
Allelic Discrimination
Detector
4. Click
Absolute or Relative
Quantification Detector
.
The software saves the new detector and displays it in the detector list.
Note: If using the SDS Enterprise Database, you cannot create a detector that
that uses the same combination of detector name, reporter dye, and quencher
dye as an existing detector.
5. Repeat steps 2 through 4 to create detectors for all remaining assays on the plate.
Note: Click the
button for information on the Detector Manager dialog box
or to view the procedures for editing, deleting, or searching for detectors.
6. Choose from the following:
If constructing a
plate document for…
3-10
Then…
• absolute quantification
• relative quantification
• dissociation curve analysis
copy the detector(s) to the plate document as
explained in the procedure on page 3-11.
allelic discrimination
create and apply markers to the plate
document as explained on page 3-12.
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Step 2 – Applying Detectors and Markers
Copying
and Applying
Detectors to the
Plate Document
IMPORTANT! Once you copy a detector to the plate document, it is no longer linked
to the corresponding entry in the Detector Manager. Consequently, if you modify a
detector using the Detector Manager after you have copied it to a plate document,
you must remove the detector from the plate document and copy it again to update
the plate document with the changes.
1. In the Detector Manager dialog box of the SDS software, copy the detectors to
the plate document:
a. While pressing and holding the Ctrl key, select the detectors you want to
apply to the plate document.
The software highlights the selected detectors.
b. Click
.
The software adds the detectors to the well inspector of the plate document.
Plate document
Copied detector
Detector Manager
Detector selected
for copying
2. Click
to close the Detector Manager.
3. In the plate grid, select the wells containing the assay for the first detector.
Note: For easier selection of plate grid wells, use the Ctrl and Shift keys to
select wells individually or in groups. See page 2-23 for more information.
4. Apply detector to the selection by clicking the check box for the detector in the
Use column of the well inspector.
Detector added to
selected wells of the
plate document
Use check box
5. Repeat steps 3 through 4 to apply the remaining detectors to the plate grid.
6. Configure the plate document with detector tasks as explained in
“Step 3 – Configuring the Plate Document with Tasks” on page 3-14.
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3-11
Chapter 3 Preparing a Run
Creating Markers
for Allelic
Discrimination
Allelic discrimination plate documents feature the use of ‘markers’ to aid in
organizing and applying detectors based on the loci they target. A marker is a pairing
of two detectors representing chemical assays designed to discriminate between
different alleles of a common locus. The SDS software uses marker information
during data analysis to organize and compare the processed run data.
IMPORTANT! Allelic discrimination plate documents must contain at least one
marker.
1. If the Detector Manager dialog box is open, click
to close it.
2. Select Tools > Marker Manager.
3. In the Marker Manager dialog box, click
.
4. In the Enter name of new Marker field of the Add Marker dialog box, enter a
name for the new marker, and click
.
The new marker appears in the Markers field.
Note: If using the SDS Enterprise Database, you cannot create a marker that
that uses the same combination of marker name and detectors as an existing
marker.
5. Apply detectors to the new marker:
a. In the Markers field, click the new marker to select it.
The software highlights the selected marker.
b. In the Detectors list, select the Use check boxes of the detectors that you
want to assign to the marker.
The software highlights the selected detector.
IMPORTANT! You cannot assign more than two detectors to a marker and the
detectors cannot use the same reporter dye.
Detectors assigned
to ‘CYP 2C9*2’
6. If evaluating multiple loci, repeat steps 3 through 5 to create additional markers
as needed.
IMPORTANT! You must configure a marker with two detectors before you can
apply it to a plate document.
Note: Click
for information on the features of the Marker Manager dialog
box or to delete markers from the Markers list.
7. Apply the marker(s) to the allelic discrimination plate document as explained in
“Copying and Applying Markers” on page 3-13.
3-12
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Step 2 – Applying Detectors and Markers
Copying and
Applying Markers
IMPORTANT! Once you copy a marker to the plate document, it is no longer linked to
the corresponding entry in the Marker Manager. Consequently, if you modify a marker
using the Marker Manager after you have copied it to a plate document, you must
remove the marker and copy it again to update the plate document with the changes.
1. In the Marker Manager dialog box of the SDS software, copy the allelic
discrimination marker to the plate document:
a. While pressing and holding Ctrl, select the marker(s) you want to apply to
the plate document.
b. Click
to copy the markers to the plate document.
c. In the Copy Markers To Plate dialog box, click
.
Markers copied to
the plate document
d. Repeat steps a and c to copy additional markers to the allelic
discrimination plate document as needed.
2. Click
to close the Marker Manager dialog box.
3. Select the wells containing the assays for a marker you configured in the
previous procedure.
Note: For easier selection of plate grid wells, use the Ctrl and Shift keys to
select wells individually or in groups. See page 2-23 for more information.
4. In the well inspector, click the Use check box of the marker you want to add to
the selected wells.
Note: The detectors associated with the marker are automatically applied to the
selected wells when the marker is placed in Use.
Detectors for the
‘PDAR CYP 2C9*2’
marker
‘PDAR CYP 2C9*2’
added to selected
wells of the plate
document
Use check box for
‘PDAR CYP 2C9*2’
(selected)
5. If necessary, repeat steps 3 through 4 to assign any remaining markers to the
plate document.
6. Configure the plate document with detector tasks as explained in
“Step 3 – Configuring the Plate Document with Tasks” on page 3-14.
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Chapter 3 Preparing a Run
Step 3 – Configuring the Plate Document with Tasks
User Access
Requirement
About Detector
Tasks
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to apply or modify detector task settings.
You must assign a ‘task’ to the detectors or markers applied to each well of the plate
document that defines their specific purpose or function on the plate. The SDS
software uses the detector task assignments to determine how to treat the data
produced by the wells when analyzing the run data. Detector tasks vary depending on
the type plate document.
Applying
Detector Tasks
1. Using the Ctrl and Shift keys, select the wells of the plate grid containing
samples for a particular task described in the table below.
Table 3-1
Detector Tasks of the SDS Software
Experiment
Allelic
Discrimination
Absolute
Quantification
Relative
Quantification
Task
Unknown
Apply to…
all detectors of wells that contain PCR reagents
and test samples.
NTC
all detectors of negative control wells that contain
reagents for the PCR, but lack template.
Unknown
all detectors of wells containing PCR reagents and
test samples for quantification.
Standard
the appropriate detectors of wells that contain
PCR reagents and samples of known quantities.
(See page 3-15 for instructions.)
NTC
all detectors of negative control wells that contain
PCR reagents, but lack template.
Target
all detectors of wells that contain PCR reagents for
the amplification of target sequences.
Endogenous
Control
all detectors of wells that contain reagents for the
amplification of the endogenous control sequence.
2. In the well inspector, click the field in the Task column for each detector entry,
and select the appropriate task from the drop-down list. The SDS software labels
all selected wells with the task.
Selected wells
(task applied
to selection)
3-14
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Task drop-down list
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Step 3 – Configuring the Plate Document with Tasks
3. Repeat steps 1 through 2 to apply any remaining tasks to the plate document.
4. Choose from the following options:
If constructing a
plate document for…
Assigning
Standards for
Absolute
Quantification
Then…
absolute quantification
assign quantities to the standard wells of the plate
document as explained below.
• allelic discrimination
• relative quantification
• dissociation curve analysis
• If necessary, set the passive reference for the
plate document as explained on page 3-16.
• Otherwise, program the method for your run as
explained on page 3-17.
For the SDS software to create a standard curve for quantification of unknown
samples, you must apply the Standard task and quantity values to your absolute
quantification plate document.
1. In the plate grid of the SDS software, select the replicate wells containing the
first standard in the dilution series.
2. In the well inspector, click the field in the Task column for the appropriate
detector entry, and select the Standard from the drop-down list.
3. In the well inspector, click the field in the Quantity column for the appropriate
detector, enter a quantity or concentration for the standard in the appropriate
unit of measurement (starting copy number, picograms, nanograms, etc.), and
press Enter.
The software labels the selected standard wells with the specified quantity.
Selected wells
(quantity applied
to selection)
Quantity field
4. Repeat steps 1 through 3 to configure the plate with the other sets of replicate
standard wells on the plate.
When finished, the plate grid should contain a complete set of replicate wells
labeled with the Standard task and assigned quantities that the software will
use to compute the standard curve for the run.
5. Do one of the following:
• If necessary, set the passive reference for the plate document as explained on
“Step 4 – Setting the Passive Reference and Omitting Wells” on page 3-16.
• Otherwise, program the method for your run as explained in
“Step 5 – Programming the Plate Document Method” on page 3-17.
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Chapter 3 Preparing a Run
Step 4 – Setting the Passive Reference and Omitting Wells
User Access
Requirement
Setting
the Passive
Reference
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to change the passive reference or omit wells from use.
If using an Applied Biosystems chemistry, use the default Passive Reference setting
(Applied Biosystems chemistries use the ROX™ passive reference dye molecule). If
running a custom chemistry, select a dye to use as a passive reference for the run.
Note: Applied Biosystems recommends using a passive reference to normalize the
signals from the reporter dyes.
1. In the Passive Reference drop-down list, select the appropriate reference dye.
Passive Reference drop-down list
Select the appropriate
passive reference
2. If necessary, omit wells from use as explained below. Otherwise, program the
method for the run as explained on page 3-17.
Omitting Wells
from Use
The SDS software allows you to omit wells from use before or after running a plate.
IMPORTANT! If you remove a well removed from use before you run a plate
document, the SDS software will not collect data for the well during the run.
However, if you remove a well from use after you run a plate document, the software
excludes the well from the analysis but does not delete the data.
1. While pressing and holding the Ctrl key, select the well(s) in the grid that you
want to remove.
2. In the well inspector of the Setup tab, click the Omit Well(s) check box.
Selected wells
(removed from use)
Omit Well(s) check box
(selected)
3. Program the method for the run as explained on page 3-17.
3-16
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Step 5 – Programming the Plate Document Method
Step 5 – Programming the Plate Document Method
User Access
Requirement
About SDS
Methods
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to modify the method of a plate document.
During a run, the SDS software controls the instrument based on the instructions
encoded in the method of the plate document. Each new plate document
(except allelic discrimination) contains a default method that you must customize for
your experiment.
Methods contain the:
•
•
•
•
•
Thermal Cycler Conditions
Auto Increment Values
Ramp Rates
Data Collection Options
Reaction Volume Setting
To create a method for…
Then…
• absolute quantification
• relative quantification
program the method as explained on page 3-19.
IMPORTANT! If using the SDS Enterprise Database, read
the discussion of considerations below.
allelic discrimination
see page 3-18.
dissociation curve analysis
To perform a dissociation curve analysis do the following:
1. Program the method for the absolute quantification
experiment as explained on page 3-19.
2. Add a temperature ramp to the method for dissociation
curve analysis as explained on page 3-21.
Database
Considerations
Configure the method of your plate document to collect data only in the necessary
stages of the PCR. Because of memory constraints, the SDS Enterprise Database
cannot save real-time plate documents greater than 40 MB. The file size for a
real-time run depends on the length of the run and the configuration and placement
of the data collection icons. Longer runs and runs configured to collect data at
multiple stages of the method can be considerably larger than the 15–25 MB average.
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Chapter 3 Preparing a Run
Programming
Methods for Allelic
Discrimination
To maximize instrument throughput, the SDS software does not provide the option to
thermal cycle allelic discrimination plate documents. Because allelic discrimination
experiments are end-point runs that do not require data collection during the PCR,
you can perform thermal cycling on a dedicated thermal cycler and then transfer to
the 7900HT instrument for data collection and analysis.
If you want to thermal cycle the
allelic discrimination plates on…
Then…
a designated thermal cycler
go on to “Step 8 – Applying Sample and Plate
Information” on page 3-25
the 7900HT instrument
follow the procedure below.
Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument
To perform the thermal cycling and the plate read using the 7900HT instrument, run
the plate first as a real-time plate document and then again as an allelic
discrimination plate document as explained below.
IMPORTANT! Follow the procedure below only if you intend to perform the PCR on
the 7900HT instrument. Otherwise, perform the PCR on a dedicated thermal cycler
and then transfer the plate to the 7900HT instrument for data collection.
1. Start the SDS software.
2. Create a real-time plate document for absolute quantification as described on
page 3-8.
Note: It is not necessary to configure the plate document with detectors.
3. Program the plate document method with the thermal cycling times and
temperatures for your protocol as described in “Programming Methods for
Absolute or Relative Quantification” on page 3-19.
4. Save the plate document as explained on page 4-24.
IMPORTANT! Do not save the real-time plate document to the database. The
database cannot store plate documents of different run types with the same bar
code.
5. Run the plate using the real-time plate document as described on page 4-25.
Note: Although large, the real-time file may be helpful in diagnosing and
troubleshooting the experiment later if the data from the allelic discrimination
run produces unexpected results.
6. When the run is complete, close the absolute quantification plate document.
7. Go on to “Step 6 – Saving the Plate Document as a Template” on page 3-22.
3-18
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Step 5 – Programming the Plate Document Method
Programming
Methods for
Absolute or
Relative
Quantification
Note: The following procedure describes how to configure only the basic features of
the method: thermal cycler conditions, sample volume, and data collection options.
To further customize the method for the plate document, click
in the Instrument
tab and refer to the online help for instructions on configuring the auto increment and
ramp rate values.
1. In the SDS software, select the Instrument tab of the plate document.
2. If necessary, select or de-select the 9600 Emulation check box.
Note: When the 9600 Emulation check box is checked, the SDS software
reduces the ramp rate of the 7900HT instrument to match that of the
ABI PRISM® 7700 Sequence Detection System instrument.
3. Modify the default thermal profile for the method as needed:
To…
Then…
adjust step
parameters
(time/temp)
select a field value, enter a new value, and click anywhere
outside of the field.
Temperature field
(4 to 99.9 °C)
Time field
(0:01 to 98:59 min)
add a hold, cycle
set, or step
1. Click the step to the left of the location you want to place the
new stage.
2. Click
or
.
The software inserts the stage into the thermal profile.
Note: To add a step to a stage, select the step to the left of the
location you want to place the step and click
.
Selected step
New step
appears here
remove a step
1. Click the step you want to remove.
The software highlights the selected step.
2. Click
to remove the step from the profile.
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3-19
Chapter 3 Preparing a Run
4. Configure the data collection options for the method:
a. Select the Data Collection tab.
b. Click below each plateau or ramp inside the cycle stage of the thermal
profile to place a data collection icon at each step.
Data collection icons
IMPORTANT! Configure the method of your plate document to collect data only
in the necessary stages of the PCR. The file size for a real-time run depends on
the length of the run and the configuration and placement of the data collection
icons. Longer runs and runs configured to collect data at multiple stages of the
method can be considerably larger than the 15–25 MB average.
5. Click the Sample Volume (µL) field and enter the volume of the reactions on
the plate.
Note: Sample volume refers to the entire contents of any well, including buffer
blank, or any combination of master mix and nucleic acids.
IMPORTANT! All wells on one plate must contain the same reaction volume.
6. Choose from the following options:
3-20
If performing an…
Then…
absolute or relative
quantification run only
go to “Step 6 – Saving the Plate Document as a
Template” on page 3-22.
absolute quantification run
with a dissociation curve
add a temperature ramp to generate dissociation
curve data as explained on page 3-21.
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Step 5 – Programming the Plate Document Method
Programming a
Temperature
Ramp for
Dissociation
Curve Analysis
To generate the data required to perform a dissociation curve analysis, the 7900HT
instrument must be programmed to run a ‘temperature ramp’ in which it slowly
elevates the temperature of the samples while collecting fluorescence measurements
once every 7-10 seconds (see page 6-38 for a detailed explanation).
1. In the Instrument tab of the plate document, select the Thermal Profile tab.
2. Click the step to the left of the location you want to place the new stage.
3. In the Thermal Profile tab, click
.
The SDS software inserts a temperature ramp at the end of the thermal profile
consisting of a set of default steps.
4. You can customize the default temperature ramp, however Applied Biosystems
recommends that you observe the following guidelines to ensure the maximum
resolution for the run (the greatest separation of the derivative peaks during the
analysis).
Guideline
Example
The Start and End steps of
the temperature ramp must:
• be separated by a
minimum temperature
difference of 35 °C.
• elapse 15 seconds (0:15)
each.
End step
Start step
The ramp rate setting for the
End step of the temperature
ramp must be 2 %.
IMPORTANT! You must
deselect the 9600 Emulation
check box to modify the
Ramp Rate Settings.
End step
ramp rate
setting
The temperature ramp in the
thermal profile must contain
a data collection icon.
Data
collection
icon
5. Go to “Step 6 – Saving the Plate Document as a Template” on page 3-22.
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3-21
Chapter 3 Preparing a Run
Step 6 – Saving the Plate Document as a Template
User Access
Requirement
Adjusting the
Display Settings
(Optional)
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save a plate document as a plate document template.
Because plate documents created from the plate document template will retain its
display settings, configure the display settings of the plate document template as you
would like the child plate documents to be displayed.
1. Click
(select View > Display Setting).
2. In the Display Settings dialog box, configure the display settings for
the Results Grid, and the Results Table.
For more information on the Display Settings dialog box or to view the
procedures for configuring the display settings for the plate document template,
click
to open the Sequence Detection Systems Software Online Help.
3. When finished, click
.
The SDS software applies the new display settings to the plate document.
4. Go to “Saving the Plate Document as a Template (Optional)” on page 3-23.
3-22
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Step 6 – Saving the Plate Document as a Template
Saving the Plate
Document as a
Template
(Optional)
IMPORTANT! Saving the plate document as a plate document template is an optional
step and recommended for instances where the document can be used to create
duplicate plate documents for a series of plates with identical assay configurations. If
you choose not to use your plate document as a plate document template, go to
“Configuring the Plate Document Information (Optional)” on page 3-25.
To save the plate document as an SDS 7900HT Template Document:
1. Select File > Save As.
2. In the Save in field of the Save As dialog box, navigate to the
AppliedBiosystems > SDS2.2.1 > Templates directory.
Note: By saving the file to the Templates directory, it becomes available from
the Template drop-down list in the New Document dialog box.
3. Select File of type > SDS 7900HT Template Document (*.sdt).
4. Click the File name field, and enter a name for the plate document template.
5. Click
.
The software saves the plate document template.
6. If the software displays one or more warnings, read the note and click
.
7. Select File > Close.
If the software prompts you to save the plate document, click
.
The SDS software closes the plate document template.
8. Create a plate document from the plate document template as explained on
page 3-24.
To save the plate document template to the SDS Enterprise Database:
1. Select File > Save Template to Database.
2. In the Save Document as Template dialog box, click
.
3. If the software displays one or more warnings, read the note and click
.
4. In the Template Name field of the Save Template to Database dialog box, enter
a name for the plate document template (up to 128-characters), and click
.
5. Select File > Close.
If the software prompts you to save the plate document, click
.
The SDS software closes the plate document template.
6. Create a plate document from the plate document template as explained on
page 3-24.
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3-23
Chapter 3 Preparing a Run
Step 7 – Creating a Plate Document from the Template
User Access
Requirement
Options for
Creating Plate
Documents from
the Template
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to create a plate document from a plate document
template.
The SDS software offers two options for creating plate documents from a plate
document template: individually or in batches.
Option
Description
See Page
Create an individual plate
document from the plate
document template
The procedure below explains how to
create a single plate document from a
plate document template for running a
plate. By repeating the procedure, you
can create as many plate documents as
needed.
Follow the
procedure
below.
Create multiple plate documents
using the Template Batch utility
As a faster alternative to the option
above, the software includes a
Template Batch utility that can
simultaneously create multiple plate
documents from the plate document
template.
IMPORTANT! If using the
SDS Enterprise Database, you
must choose this option.
Creating a SDS
Plate Document
from a Template
1. In the SDS software, click
4-36
(or select File > New).
2. Configure the New Document dialog box:
• Assay drop-down list – Select the same assay as the plate document
template.
• Container drop-down list – Select the same plate type as the plate
document template.
• Template drop-down list – Select the plate document template (*.sdt)
created on page 3-22.
If the plate document template does not appear inside the Template drop-down
list, select the file as explained below:
a. Click
.
b. In the Look in field of the Open dialog box, navigate to and select the plate
document template (*.sdt) created on page 3-22.
c. Click
.
The Template drop-down list displays the plate document template.
Note: The Template drop-down list displays all plate document templates
contained in the Templates subdirectory of the SDS program directory.
• Barcode field – (Optional) Do one of the following:
– Enter the bar code number for the plate, or
– Scan the plate bar code using the hand-held bar code scanner.
3. Click
to create a new plate document from the plate document template.
4. Configure the new plate document with sample names and plate information as
explained on page 3-25.
3-24
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Step 8 – Applying Sample and Plate Information
Step 8 – Applying Sample and Plate Information
User Access
Requirement
Applying Sample
Names to the
Plate Document
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to apply (or modify) sample names to the plate document.
The plate document must contain sample attributes to effectively organize and
analyze data produced from the run. Once applied, the software displays the sample
names in the plate grid and table views.
Note: You can apply sample names after the plate has been run, but they must be
added before the analysis of the run data.
Note: The SDS software provides the ability to import setup table information
(detector, detector task, and sample name layouts) into a plate document from a
tab-delimited text file. See “Importing Plate Document Setup Table Files” on
page A-2 for more information.
1. In the plate grid, select the wells containing the first sample.
2. Click the Sample Name field, enter a name for the sample, and press Enter.
The software labels the selected wells with the new sample name.
3. Repeat steps 1 through 2 for all remaining samples.
Sample name
appears here
Sample Name field
4. Configure the plate document with plate information as explained below.
Configuring the
Plate Document
Information
(Optional)
1. In the SDS software, select Tools > Document Information.
2. In the Document Information dialog box, edit the Barcode, Operator, or
Plate Comments information.
IMPORTANT! If using an SDS Enterprise Database:
• The bar code entered into the plate document must be unique and cannot be
used in another plate document.
• The software automatically populates the Operator field with the name of the
user who logged into the SDS software and performed the data collection.
The software populates the field only after the plate document has been run.
• Do not modify the bar code of a plate document that contains data from a run.
3. When finished, click
.
4. Run the plate document and associated plate as explained on page 3-26.
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3-25
Chapter 3 Preparing a Run
Step 9 – Running the Plate on the 7900HT Instrument
User Access
Requirement
There is no access requirement. All users can run plates that have been saved to the
SDS Enterprise Database.
IMPORTANT! A plate document must be saved to the database before a user
belonging to the Operator User Group can run it.
Options
for Running
SDS Plates
The 7900HT instrument can run prepared plates individually or in groups using the
Zymark Twister Microplate Handler.
IMPORTANT! If you are not using a Zymark Twister Microplate Handler, you must
run plates individually.
Choose one of the following options to run the plate:
To run the plate…
Description
individually
1. Prepare the consumable (optical plate or
TaqMan® Low Density Array)
4-5
2. Run the plate/array individually using the SDS
software.
4-23
1. Prepare the consumables (optical plates or
TaqMan® Low Density Arrays)
4-5
2. Run the plate/array with others as part of a batch
from the Automation Controller Software using
the Zymark Twister Microplate Handler.
4-31
as part of a group
(Automated Operation)
See Page
IMPORTANT! You must have a Zymark Twister
Microplate Handler to run plates using this option.
3-26
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Operating the Instrument
In This Chapter
4
4
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
Section 4.1 Consumable Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Preparing TaqMan Low Density Arrays for Use. . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
Section 4.2 Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23
Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24
Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . 4-25
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29
Section 4.3 Automated Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-31
Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32
Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34
Running Plates Using the Automation Controller Software . . . . . . . . . . . . . . . . . 4-41
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44
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4-1
Chapter 4 Operating the Instrument
Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when creating and
modifying plate documents as described in this chapter. This section describes the
types of actions you may need to perform depending on how your administrator has
configured the database. For more information on any of the features described
below, see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast
Real-Time PCR System Administrators Guide.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
When the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 4-1
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
When the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 4-2
4-2
Electronic Signature Verification Dialog Box Options
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Before You Begin
Before You Begin
Pre-Run
Checklist
The following tasks must be complete to run plates on the 7900HT instrument. See
the associated page number for details on each procedure.
Done
Check
See Page
A background run has been performed in the last month
7-16
A pure dye run has been performed in the last 6 months
7-20
The instrument tray does not contain a plate
4-44
IMPORTANT! The instrument tray must be empty to begin a run. If
the instrument tray contains a plate, you must eject and remove it.
If using an Automation Accessory, also check the following…
The output stack does not contain plates.
Workflow
Overview
–
1. Prepare your experiments for use on the 7900HT instrument:
– Prepare optical plate(s) (see page 4-8).
– Prepare TaqMan® Low Density Array(s) (see page 4-10).
2. Prepare and run the 7900HT instrument:
– Stand-alone Operation (see page 4-23).
– Automated Operation using the SDS Enterprise Database (see page 4-32).
– Automated Operation without the database (see page 4-34).
3. Analyze the run data.
– Allelic Discrimination Analysis (see page 5-5).
– Absolute Quantification Analysis (see page 6-5).
– Relative Quantification Analysis (see page 6-15).
– Dissociation Curve Analysis (see page 6-37).
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Chapter 4 Operating the Instrument
4-4
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Section 4.1 Consumable Preparation
Section 4.1 Consumable Preparation
In This Section
Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Preparing TaqMan Low Density Arrays for Use. . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
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4-5
Chapter 4 Operating the Instrument
Preventing Contamination
Contamination
and the
5´ Nuclease
Assay
General PCR
Practices
PCR using the 5´ nuclease assay requires special laboratory practices to avoid false
positive amplifications (Kwok and Higuchi, 1989). The assay’s logarithmic
amplifications can potentially amplify a single DNA molecule (Saiki et al., 1985;
Mullis and Faloona, 1987).
Observe the following guidelines when assembling optical plates or loading
TaqMan® Low Density Arrays for use on the 7900HT instrument:
• Wear a clean lab coat (not previously worn while handling amplified PCR products
or used during sample preparation) and clean gloves when preparing samples for
PCR amplification.
• Change gloves whenever you suspect they are contaminated.
• Maintain separate areas, dedicated equipment, and supplies for:
– Sample preparation
– PCR setup
– PCR amplification
– Analysis of PCR products
• Never bring amplified PCR products into the PCR setup area.
• Open and close all sample tubes carefully. Try not to splash or spray PCR samples.
• Keep reactions and components capped as much as possible.
• Use positive-displacement pipets or aerosol-resistant pipet tips.
• Clean lab benches and equipment with 10% bleach solution.
Compatible Consumables
Consumables for
Use with
the 7900HT
Instrument
Table 4-1
Compatible Consumables
Compatible Seals
Consumable Type
ABI PRISM™
384-Well Optical
Reaction Plates
4-6
Use these…
Not these…
• ABI PRISM™ Optical Adhesive Covers
(quantity 100)
• Optical Adhesive Covers (quantity 25)
384-Well Septa
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Compatible Consumables
Table 4-1
Compatible Consumables
Compatible Seals
Consumable Type
Use these…
Not these…
ABI PRISM™ 96-Well
Optical Reaction
Plates
• Compression Pads:
– Standard pad for manual operation
– ABI PRISM™ Snap-On Compression
Pad (reusable)
• 96-Well Plate Seals:
– ABI PRISM™ Optical Adhesive Covers
(quantity 100)
– Optical Adhesive Covers
(quantity 25)
– ABI PRISM™ Optical Caps
(flat caps only)
• ABI PRISM™
Optical Caps
(round caps)
• 96-Well Septa
Optical 96-Well
Fast Thermal
Cycling Plates
• ABI PRISM™ Optical Adhesive Covers
(quantity 100)
• Optical Adhesive Covers (quantity 25)
TaqMan® Low
Density Arrays
None (self sealing)
None (self sealing)
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4-7
Chapter 4 Operating the Instrument
Preparing Optical Plates for Use
Preparing Plates
for Use on the
7900HT
Instrument
1. Prepare the reactions in an optical plate by aliquoting reagents, enzyme, and
samples to the appropriate wells of an optical plate.
IMPORTANT! The arrangement of the reactions (samples and assays) on the
plate must match the configuration of information (sample names and
detectors/markers) on the corresponding plate document.
2. Seal the optical plate with a seal that is compatible with the 7900HT instrument
(Figures 4-3 and 4-4 on page 4-9).
Note: See “Compatible Consumables” on page 4-6 for a list of seals for use
with the Applied Biosystems 7900HT Fast Real-Time PCR System.
3. Briefly centrifuge the plate to collect the reactions at the bottom of the wells and
to eliminate any air bubbles that may be present.
4. Visually verify that each reaction is positioned at the bottom of its well.
Correct Position
The sample is positioned
correctly in the bottom of
the well.
Incorrect Position
The sample lies on the
side wall because the
plate was not centrifuged.
An air bubble lies at the
bottom of the well
because the plate was
not:
• Centrifuged with enough force, or
• Centrifuged for enough time
5. If running an ABI PRISM 96-Well Optical Reaction Plate, apply the appropriate
compression pad to the sealed optical plate.
If you are going to run the plate:
• Individually using the SDS software, then apply a standard compression
pad (Figure 4-3)
• As part of a batch using the Automation Controller Software, then apply an
ABI PRISM Snap-On Compression Pad (Figure 4-3)
IMPORTANT! The ABI PRISM™ Snap-On Compression Pad/plate assembly
may still be hot after the Zymark® Twister Microplate Handler loads it into
the Plate Handler’s output stack. Please wait at least 30 seconds before
manually handling the Snap-On Compression Pad/plate assembly.
6. Choose from the following. If performing:
– Absolute or Relative Quantification, or Dissociation Curve Analysis
Run the plate as explained in “Running the Plate” on page 4-25.
– Allelic Discrimination
4-8
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Preparing Optical Plates for Use
a. Load the plate onto a designated thermal cycler and perform the PCR.
b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells and
to eliminate any air bubbles that may have formed during thermal cycling.
c. Run the plate as explained in “Running the Plate” on page 4-25.
Standard compression
pad (for single-plate
runs)
‘Snap-On’
Compression Pad
(for automated runs)
Optical Caps
(flat caps only)
Optical adhesive
cover
OR
Figure 4-3
96-Well Optical Reaction Plate Assembly
Optical adhesive
cover
384-well
plate
Figure 4-4
384-Well Optical Reaction Plate Assembly
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4-9
Chapter 4 Operating the Instrument
Preparing TaqMan Low Density Arrays for Use
About the Low Density Arrays
The TaqMan Low Density Arrays are research tools for profiling gene expression
using the Comparative CT Method of relative quantification. The Low Density Array
evaluates from one to eight cDNA samples or controls generated from human total
RNA in a two-step RT-PCR experiment.
How It Works
The Low Density Array functions as an array of reaction vessels for the
PCR/sequence detection step. Typically, the wells of the Low Density Array contain
Applied Biosystems fluorogenic 5´ nuclease assays (TaqMan® reagents; see the note
below) that detect the real-time amplification of user-specified targets. Relative
levels of gene expression are determined from the fluorescence data generated during
PCR using the Applied Biosystems 7900HT Fast Real-Time PCR System Relative
Quantification software.
Note: In this document, the term TaqMan reagents refers to 5´ exonuclease assay
reagents that feature Applied Biosystems TaqMan® primers and probes (includes
Applied Biosystems PDARs, TaqMan® SNP Genotyping and Gene Expression
Assays, Custom TaqMan® SNP Genotyping and Gene Expression Assays, and any
custom TaqMan reagents.)
Low Density
Array
Components
The Low Density Array (Figure 4-5) acts as a vessel for real-time PCR. It consists of
a series of 384 interconnected wells divided into eight sets of assays. Each set
provides a user-specified number of replicates. Each well contains dried Applied
Biosystems TaqMan primers and probes for one mRNA target.
Fill reservoir (1 of 8)
A reservoir for the cDNA sample or
control before it is centrifugally
loaded into the wells
Fill consumable
The segment of the Low Density
Array that contains the eight fill
reservoirs. (After the Low Density
Array is centrifuged and sealed, the
fill consumable is trimmed off.)
GR2156a
Figure 4-5
4-10
Barcode Provides coded access to
Low Density Array configuration
databases
The TaqMan Low Density Array
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Preparing TaqMan Low Density Arrays for Use
Internal Structure
Figure 4-6 illustrates the Low Density Array internal structure.
Fill port
Vent port
Fill reservoir
Main
Feeder channel
Reaction well
Figure 4-6
Detectors
TaqMan Low Density Array, Internal Structure
The Low Density Array permits the amplification of target and an external
endogenous control cDNA using fluorogenic 5´ nuclease assays. The assays consist
of two reactions, each a complete PCR system with corresponding probe and
primers.
The fluorogenic probes of the assays function as follows:
• Probes labeled with the FAM™ dye detect the amplification of user-specified
cDNA targets.
• Probes labeled with the FAM™ dye detect the amplification of the external
endogenous control.
Genomic DNA
Contamination
TaqMan probes and primers for the cDNA targets span exon-exon junctions to
minimize the contribution of contaminating genomic DNA. Performance tests
demonstrate that 5´ exonuclease assays can be run with samples containing up to
10,000 copies of genomic DNA without detection of contaminants, unless otherwise
specified by Applied Biosystems.
External Endogenous Control Assay
Note that the commonly-used external endogenous control assay is not RNA-specific
and consequently is affected by genomic DNA contamination. However, because of
the extremely high expression level of rRNA, even gross DNA contamination has a
negligible effect on the relative quantification values obtained from the Low Density
Array.
Single-Plex
Reactions
The Low Density Array is recommended for use with single-plex TaqMan reagents
(for example, FAM dyes). An external endogenous control assay (for example,
FAM-18S or FAM-GAPDH) is used as a calibrator in relative quantification
calculations.
Note: For more information on relative quantification, see ABI PRISM ® 7700
Sequence Detection System User Bulletin #2: Relative Quantitation of Gene
Expression (PN 4303859).
Quality Control
Functional verification of the preloaded probes and primers inside the Low Density
Array is performed as part of the Applied Biosystems manufacturing quality control
process.
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Chapter 4 Operating the Instrument
About the Centrifuge System
After the fill reservoirs of a Low Density Array have been loaded with the cDNA
sample or control, the centrifuge system is required to distribute the cDNA sample or
control to the reaction wells. The centrifuge system supports the simultaneous
loading of up to 12 Low Density Arrays.
Required
Centrifuge and
Accessories
To properly centrifuge the Low Density Arrays, you will need the following
centrifuge and centrifuge accessories:
• Sorvall® Legend T Centrifuge
GR2170
Figure 4-7
Sorvall Legend T Centrifuge
• Sorvall®/Heraeus “Tool-less” 750-mL Swing-Out Rotor Body
• A set of Sorvall®/Heraeus Custom Buckets (four), each with its own card holder
(Contact your Applied Biosystems representative to order the custom buckets.)
• Current firmware
If you are upgrading an existing Sorvall Legend T or Legend RT Centrifuge,
you may also need to upgrade the firmware so that it can recognize the Low
Density Array bucket type.
Centrifuge
System
Components
Figure 4-8 illustrates the centrifuge system components.
Centrifuge rotor
Provides the rotational drive for
a set of four centrifuge buckets
GR2154
Card holder
Supports the Low Density
Array’s fill reservoirs during
centrifugation
Figure 4-8
4-12
GR2153
GR2152
Bucket
Contains the Low Density
Arrays and card holder
during centrifugation
Centrifuge System Components
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Preparing TaqMan Low Density Arrays for Use
Laboratory Setup
Setting the
Bucket Type for
the Low Density
Array.
The centrifuge system is designed to be placed on a standard laboratory bench. It
requires access to 115 VAC, 60 Hz electrical power. Consult the owner’s manual
should for specific operating and site preparation instructions.
1. Power on the centrifuge.
2. Following the instructions in the Sorvall Legend T Centrifuge operator’s
manual, use the front panel controls on the centrifuge to set the bucket type for
the centrifuge to 15679
Bucket type control
Scrolling control
IMPORTANT! Be sure to set the correct bucket type. Setting the correct bucket
type ensures that the maximum rotational speed stays within the manufacturer’s
specified limits.
3. Using the front panel controls, set the following operational parameters:
Parameter
Value
Up ramp rate
9
Down ramp rate
9
Rotational speed
1200 rpm (331 × g)
Centrifugation time
1 min
rpm indicator
Buttons 1–4 are
programming
buttons.
Up ramp
rate control
Down ramp
rate control
rpm
controls
Time
controls
4. Power off the centrifuge.
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Chapter 4 Operating the Instrument
About the Sealer
The microfluidic card sealer is required to isolate the wells of the Low Density Array
after it is loaded with cDNA samples or controls and master mix. The sealer uses a
precision stylus assembly (carriage) to seal the main fluid distribution channels of the
Low Density Array.
Sealer
Components
Handle
Used to move the carriage,
which contains the stylus
assembly
Low Density Array
Shown foil-side up, with fill reservoirs on the right
(carriage moves from left to right during sealing, in
the direction of the arrows etched into the base)
GR2172
Insert plate
Holds the Low Density
Array during sealing
Figure 4-9
Laboratory Setup
Base
Provides a stable platform for
the Low Density Array and a
track for the stylus assembly
Sealer Components (Shown with Cover Removed)
The microfluidic card sealer is designed to be placed on a standard laboratory bench.
It requires no electrical power.
Loading the TaqMan Low Density Arrays
Equipment and
Materials Needed
You need the following equipment and materials to load the sample-specific PCR
reaction mix into the fill reservoirs of the Low Density Arrays:
•
•
•
•
Guidelines for
Loading the
Cards
Rainin F-100 micropipette (100-µL)
Rainin Fine Point pipet tips (100-µL)
TaqMan Low Density Array(s)
Sample-specific PCR reaction mix (see the protocol accompanying your Low
Density Array chemistry for instructions on preparing the reaction mix)
Follow the guidelines below to ensure proper loading of the Low Density Arrays.
• Do not remove a Low Density Array from its packaging until the packaging has
reached room temperature and you are ready to load it with the sample-specific
PCR reaction mix.
IMPORTANT! Prolonged exposure to indoor lighting can photo-degrade the
fluorescent probes contained inside the Low Density Array. Do not expose the
Low Density Array to sunlight.
• Do not twist or bend the fill consumable portion of the Low Density Array
(Figure 4-5 on page 4-10) before loading.
IMPORTANT! To successfully load the sample-specific PCR reaction mix, it is
critical that the main channels leading from the Low Density Array’s fill
reservoirs to its wells are open and undamaged.
4-14
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Preparing TaqMan Low Density Arrays for Use
Loading the Fill
Reservoirs with
Sample-Specific
PCR Reaction
Mix
1. When the packaging has reached room temperature and you are ready to load
PCR reaction mix, carefully remove a Low Density Array from its packaging.
2. Place the Low Density Array on a lab bench, with the foil side down.
3. Load 100 µL of the desired sample-specific PCR reaction mix into a 100-µL
micropipette.
4. Hold the micropipette in an angled position and place the tip in the fill port.
Note: There is a fill port on the left arm of each fill reservoir; it is the larger of
the two holes.
Fill port
Vent port
GR2158
IMPORTANT! Do not allow the tip to contact and possibly damage the coated
foil beneath the fill port.
5. Dispense the sample-specific PCR reaction mix so that it sweeps in and around
the fill reservoir toward the vent port.
IMPORTANT! Pipette the entire 100 µL into the fill reservoir.
IMPORTANT! Do not allow the tip to contact and possibly damage the coated
foil beneath the fill port.
IMPORTANT! Be careful when pushing the micropipette plunger to its second
stop position (to expel the sample-specific PCR reaction mix from the tip). If a
large amount of air is released, it can push the reaction mix out of the fill
reservoir via the vent port.
GR2159
6. To transfer the sample-specific PCR reaction mix from the fill reservoirs into
the reaction wells, the Low Density Array must be centrifuged.
Continue with “Centrifuging the TaqMan Low Density Arrays” on page 4-16.
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Chapter 4 Operating the Instrument
Centrifuging the TaqMan Low Density Arrays
Equipment and
Materials Needed
You need the following equipment and materials to centrifuge the Low Density
Arrays:
• TaqMan Low Density Array(s), containing sample-specific PCR reaction mix
(page 4-14)
• Sorvall® Legend T Centrifuge
• Sorvall®/Heraeus Custom Buckets (four required), each with its own card holder
• Blank balance cards
Placing Cards in
the Buckets
Note: The centrifuge holds four Sorvall/Heraeus buckets. Each bucket holds up to
three Low Density Arrays (loaded and/or blank balance cards).
1. Obtain an empty Sorvall/Heraeus Custom Bucket and a card holder for the
centrifuge.
IMPORTANT! The Sorvall/Heraeus buckets and card holders required for the
Low Density Arrays are custom-made. Do not use any other bucket/card holder
system for this procedure.
2. Place the bucket on a lab bench, with the label facing you.
3. Insert Low Density Arrays into the card holder, making sure that:
• The fill reservoirs project upwards out of the card holder
• The reaction wells face the same direction as the “This Side Out” label
IMPORTANT! Use blank balance cards to fill any remaining positions in the
racks.
Note: The card holder supports the Low Density Array fill reservoirs during
GR2155
centrifugation.
4. Place a filled card holder in the bucket so that the “This Side Out” label faces
the front of the bucket, which has the Sorvall emblem on it.
GR2160
Sorvall
emblem here
4-16
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Preparing TaqMan Low Density Arrays for Use
Placing the
Buckets in the
Centrifuge
1. Make sure the centrifuge is powered on, but is not rotating.
2. Open the centrifuge cover by pressing the OPEN button.
OPEN cover button
3. Place a loaded bucket onto an open rotor arm of the centrifuge.
Note: Make sure the bucket can swing easily within its slotted position on the
rotor arm.
4. Place the remaining buckets onto the rotor arms, per step 3.
The manufacturer recommends running the centrifuge with
all four buckets, even if only two buckets contain Low Density Arrays.
GR2161
Make sure the buckets and their contents are balanced.
Opposing buckets should have matching weights. If the buckets are not fully
loaded with Low Density Arrays containing the sample-specific PCR reaction
mix, place blank balance cards and card holders into the buckets.
5. Close the centrifuge cover.
Centrifuging
the Cards
1. Press the START button.
START
button
The centrifuge starts, then automatically stops after 1 min, per the programmed
sequence.
2. Repeat step 1 so that the Low Density Arrays are centrifuged for a total of two
consecutive, 1-min spins to ensure complete distribution of the sample-specific
PCR reaction mix.
3. Open the centrifuge cover by pressing the OPEN button.
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Chapter 4 Operating the Instrument
4. When the cover has fully opened, remove the buckets from the centrifuge, then
remove the card holders from the buckets.
5. Remove all Low Density Arrays from the buckets by gently lifting them by their
carrier sides.
6. Examine the Low Density Arrays to determine whether filling is complete.
The amount of cDNA sample or control remaining in the fill reservoirs should
be uniform and consistent from reservoir to reservoir.
• If there is excess cDNA sample or control remaining in a fill reservoir (as
shown below), filling is incomplete. Proceed to the next step.
• If a fill reservoir is completely drained (as shown below), it is possible that
some wells were not filled properly.
– The Low Density Array should not be processed further. Use another
Low Density Array.
– Alternatively, you can process the Low Density Array further; however,
you should void the results for the affected fill reservoir (sample).
7. Evaluate the filling as follows:
If all fill reservoirs are...
Then...
not uniform after the first
centrifuge cycle
the Low Density Array may be centrifuged again in the
same manner as before for 1 additional min.
not uniform after the
additional centrifuge cycle
• The Low Density Array should not be processed
further. Use another Low Density Array.
• Alternatively, you can process the Low Density Array
further; however, you should void the results for the
affected fill reservoir (sample).
IMPORTANT! Do not exceed 1200 rpm or accumulated centrifugation times of
more than 3 min. Excessive centrifugation speeds and times may deform the
Low Density Array.
4-18
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Preparing TaqMan Low Density Arrays for Use
Sealing the TaqMan Low Density Arrays
Equipment and
Materials Needed
You need the following equipment and materials to seal the Low Density Arrays:
Guidelines for
Sealing the Cards
Follow the guidelines below to ensure the Low Density Arrays are properly sealed.
Positioning the
Sealer
• Microfluidic card sealer
• Sturdy lab bench
• TaqMan Low Density Array(s), containing sample-specific PCR reaction mix
and centrifuged (page 4-16)
• Scissors
• Seal the Low Density Arrays as soon as possible following centrifugation. The
risk of cross-contamination is minimized by the sealing process.
• Always place the Low Density Arrays into the sealer so that the sealer’s carriage
(stylus assembly) travels toward the fill reservoir end of the Low Density Array.
• Always seal in the direction indicated by the arrows etched into the base of the
sealer to ensure that the cDNA sample or control is pushed from the Low
Density Array’s channels into its fill reservoirs.
The sealer is best operated by pushing the carriage away from you in a near-to-far
motion (not in a horizontal, side-to-side motion). Follow the procedure below to
correctly position the sealer.
1. Place the sealer on a sturdy lab bench, approximately waist high so that it can be
easily used.
2. Turn the sealer so that the front end (where sealing starts) is closest to you and
the back end is farthest from you.
Note: In the correct position, the arrows on the sealer are pointing away from
you.
GR2171
Carriage
Inserting a Card
Into the Sealer
Starting
position
1. Place the sealer’s carriage in its starting position.
IMPORTANT! Never insert a Low Density Array into the sealer if the carriage is
not in its starting position. If you move the carriage across the Low Density
Array to return it to its starting position, serious damage to the Low Density
Array will occur.
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Chapter 4 Operating the Instrument
2. Insert a Low Density Array into the sealer:
a. Orient the Low Density Array in the proper direction over the sealer’s insert
plate. The Low Density Array’s fill reservoir end should be the end closest
to the arrows etched in the base of the sealer.
b. Insert the Low Density Array, foil side up, on top of the insert plate.
GR2177
3. Gently push the Low Density Array until it is seated securely in the insert plate.
Note: Two metal pins on the sealer’s insert plate project into corresponding
holes molded into the Low Density Array’s carrier to provide mechanical
alignment for the sealing process. When properly seated, the Low Density
Array’s foil surface should be level with the base of the sealer.
Note: Four spring clips ensure that the Low Density Array is held in the proper
position.
Pin
Pin
GR2174
Sealing a Card
1. Push the carriage across the base of the sealer in the direction of the arrows. Use
one firm, smooth motion until you reach the end of the sealer.
The sealer has mechanical stops at both ends to prevent the
carriage from coming off. Do not use excessive force or speed when pushing the
carriage. Using force will not increase the sealing quality but only cause wear to
the sealer.
End position
GR2176
Move in this
direction:
4-20
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Preparing TaqMan Low Density Arrays for Use
2. Remove the sealed Low Density Array by grasping its sides and lifting it off the
sealer’s insert plate.
Note: In the middle of the sealer’s insert plate, there is a thumb slot to help you
easily access one side of the Low Density Array.
Thumb slot
GR2175
3. Inspect the Low Density Array for proper sealing. The indentations from the
stylus assembly should match up with the Low Density Array’s main channels.
If the indentations do not match up or if the foil is in any way damaged, do not
use the Low Density Array.
4. Return the carriage to its starting position on the base of the sealer.
IMPORTANT! Do not move the carriage back before removing the Low Density
Array.
Trimming Off the
Fill Reservoirs
1. Using scissors, trim the fill reservoirs from the Low Density Array. Use the edge
of the Low Density Array’s carrier as a guide.
GR2168
PHYSICAL INJURY HAZARD. Take care when trimming
off the fill reservoirs. Use scissors rather than razor blades or other unprotected
cutting devices.
The Low Density Array is now ready to be run on the 7900HT instrument.
2. Choose from the following. If performing:
– Absolute or Relative Quantification, or Dissociation Curve Analysis
Run the plate as explained in “Running the Plate” on page 4-25.
– Allelic Discrimination
a. Load the plate onto a designated thermal cycler and perform the PCR.
b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells and
to eliminate any air bubbles that may have formed during thermal cycling.
c. Run the plate as explained in “Running the Plate” on page 4-25.
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Chapter 4 Operating the Instrument
4-22
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Section 4.2 Running an Individual Plate
Section 4.2 Running an Individual Plate
In This Section
Pre-Run
Checklist
Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24
Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . 4-25
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29
The following tasks must be complete to run plates on the 7900HT instrument. See
the associated page number for details on each procedure.
Done
Check
See Page
A background run has been performed in the last week
7-16
A pure dye run has been performed in the last 6 months
7-20
The instrument tray does not contain a plate
4-44
IMPORTANT! The instrument tray must be empty to begin a run. If
the instrument tray contains a plate, you must eject and remove it.
Workflow
Overview
1. Save the SDS Plate Document (see page 4-24).
2. Load and run the prepared optical plate or Low Density Array (see page 4-25).
3. Analyze the run data.
– Allelic Discrimination Analysis (see page 5-5).
– Absolute Quantification Analysis (see page 6-5).
– Relative Quantification Analysis (see page 6-15).
– Dissociation Curve Analysis (see page 6-37).
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4-23
Chapter 4 Operating the Instrument
Saving the Plate Document
User Access
Requirement
Saving the Plate
Document for
Single Plate
Operation
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save a plate document to the database.
Before the plate document can be run, you must save it to the SDS Enterprise
Database or as an SDS 7900HT Document (*.sds).
To save as an SDS 7900HT Document:
1. In the SDS software, select File > Save As.
2. In the Look in field of the Save As dialog box, navigate to and select a directory
for the software to receive the new file.
3. In the File name field either:
• Enter a file name for the plate document file, or
• Enter or scan the bar code number for the plate into the field.
Note: The SDS software does not require that the file name match the bar code
of the corresponding plate.
4. Click
.
The software saves the plate document to the specified directory.
5. Run the plate document and associated plate as explained on page 4-25.
To save to the SDS Enterprise Database:
1. Select File > Save Document to Database.
2. Configure the Save Document to Database dialog box:
a. In the Comment field, enter a brief description of the plate document
(up to 255-characters).
If saving a plate document file to the database, you will loose the file name
unless you enter it into the Comments field.
b. Click
.
3. In the Saved Document dialog box, click
.
4. Run the plate document and associated plate as explained on page 4-25.
4-24
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Running a Single Plate (Using the SDS Software)
Running a Single Plate (Using the SDS Software)
User Access
Requirement
Running the Plate
There is no access requirement. All users can run plates that have been saved to the
SDS Enterprise Database.
1. In the SDS software, select the Instrument tab of the plate document.
2. In the Real-Time or Plate-Read tab of the Instrument tab, click
.
The instrument tray rotates to the OUT position.
3. Place the prepared optical plate or Low Density Array into the instrument tray
as shown below.
Before loading the plate or Low Density Array onto the instrument tray, make
sure that:
• The associated plate document is open in the SDS software.
• The optical plate or Low Density Array has been sealed.
Well A1
Notched corner
Bar code
Well A1
Notched corner
Bar code
IMPORTANT! The A1 position is located in the top-left side of the instrument.
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4-25
Chapter 4 Operating the Instrument
4. Do one of the following:
• If performing a real-time run, click
• If performing an end-point run, click
.
.
The instrument tray rotates to the IN position and the instrument performs the
run or plate-read.
Note: Before starting the run, the instrument may pause (up to 15 minutes) to
heat the heated cover to the appropriate temperature.
Note: For more information on the elements of the Real-Time and Plate-Read
tabs, click
and see the Sequence Detection Systems Software Online Help.
The following options are available during and after the completion of the run:
To…
See Page
Monitor the progress of the run
4-27
Stop the run
4-28
IMPORTANT! If you must stop a run in-progress for any reason,
carefully read the instructions on page 4-28 before halting the run.
4-26
Open the instrument tray (after the run)
4-29
Analyze the run data after the run is complete
4-29
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Running a Single Plate (Using the SDS Software)
Monitoring
Instrument
Progress
The SDS software displays instrument status and run progress in the Real-Time tab
(real-time runs) or the Plate Read tab (end-point runs) of the respective plate
document. Figure 4-10 shows examples of the tabs during operation of the 7900HT
instrument.
Real-Time Tab
(Real-Time Runs)
Figure 4-10
Plate Read Tab
(End-Point Runs)
Real-Time and Plate Read Tabs of the SDS Software
• Status – Displays the condition of the 7900HT instrument
• Time Remaining – Displays the calculated time remaining in the run
Temperature group box (Real-Time Plate Documents Only)
• Block – Displays the actual temperature of the sample block module
• Cover – Displays the actual temperature of the heated cover
• Sample – Displays the calculated temperature of the samples
Cycle group box (Real-Time Plate Documents Only)
•
•
•
•
•
Rep – Displays the current cycle repetition
Stage – Displays the current stage of the thermal cycling
State – Displays the current condition of the cycle stage
Step – Displays the current step being run
Time – Displays the calculated time remaining in the current step
Data Collection Stamp group box (End-Point Plate Documents Only)
• Pre – Displays the date that the instrument performed the pre-read.
• Post – Displays the date that the instrument performed the post-read.
Note: A ‘pre’ read is a plate read performed before a plate has undergone thermal
cycling.
Note: A ‘post’ read is a plate read performed after a plate has undergone thermal
cycling.
Note: For more information on the elements of the Real-Time and Plate-Read tabs,
click
and see the Sequence Detection Systems Software Online Help.
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4-27
Chapter 4 Operating the Instrument
Stopping the
Run Using the
SDS Software
IMPORTANT! Read the following directions carefully before stopping a
run-in-progress.
Stopping End-Point Runs (Allelic Discrimination)
In the Plate-Read tab of the plate document, click
.
Stopping Real-Time Runs (Absolute or Relative Quantification)
Has the Cover reading in the Real-Time tab reached 104 °C?
• No (the heated cover temperature is below 104 °C)
In the Real-Time tab of the plate document, click
.
• Yes (the heated cover temperature has reached 104 °C)
The instrument is running the plate and has begun thermal cycling. Determine a
course of action from the options in the following table:
Reason for Stopping the Run
Then…
Forgot to make a change to the plate
document such as adding a detector,
detector task, or sample name
(not including mistakes to the
temperature profile)
do not stop the run.
• Forgot to add a reagent to the plate
(such as enzyme or master mix)
• Programmed the plate document
with the wrong thermal profile
1. In the Real-Time tab of the plate
document, click
.
Allow the instrument complete the run,
then edit the plate document before
analyzing the data. The software does not
use detector or sample information until
you analyze the plate document.
2. Determine how far into the run the
instrument has progressed.
3. Based on the state of the run, determine
whether the plate can be re-run.
4. If necessary, eject the plate and add the
missing reaction component.
5. If desired, re-run the plate by re-creating
the plate document.
4-28
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After the Run
After the Run
User Access
Requirement
Ejecting a Plate
(Opening and
Closing the
Instrument Tray)
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save a plate document.
View the instrument status icon to determine the plate document to use to eject the plate.
Instrument Status
Action
1. In the SDS software, select Window > (the plate document
connected to the instrument).
Connected to
platename
2. In the plate document, select the Instrument tab
3. Select the Plate-Read or Real-Time tab.
4. Click
.
1. In the SDS software, click
2. Click
Disconnected
(or select File > New).
.
3. Select the Instrument tab.
4. Select the Plate-Read or Real-Time tab.
5. Click
Saving the
Run Data
.
After the run, you must either save the results to the SDS Enterprise Database or save
the run data to the SDS 7900HT Document (*.sds).
To save the run data to the SDS 7900HT Document:
1. In the SDS software, select File > Save.
To save the run data to the SDS Enterprise Database:
IMPORTANT! If you saved the plate document to the database prior to running the
plate, the SDS software stored the run data as a temporary file in the /temp directory. If
you closed your plate document at the end of the run without saving it, open the temp
directory and rename the temporary file to *.sds and you will be able to open the file.
1. Select File > Save Document to Database:
2. In the Comment field of the Save Document to Database dialog box, enter a
brief description of the plate document (up to 255-characters).
Note: If saving a plate document file to the database, you will loose the file
name unless you enter it into the Comments field.
3. Click
.
4. In the Saved Document dialog box, click
Analyzing the
Run Data
.
Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5
Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5
Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15
Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37
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4-29
Chapter 4 Operating the Instrument
Disconnecting
the Software from
the Instrument
The SDS software has the ability to halt all communications with the 7900HT
instrument. The ‘disconnect’ option permits the simultaneous operation of both the
SDS software and the Automation Controller Software. Because both programs
control the Applied Biosystems 7900HT Fast Real-Time PCR System, one program
must relinquish control of the 7900HT instrument before the other can be used to
operate it.
To…
Then…
disconnect the
software from
the instrument
1. In the SDS software, select the Instrument tab of the open
plate document.
2. Select the Real-Time or Plate-Read tab.
3. Click
.
Note: Once disconnected, the software neither monitors nor
controls the 7900HT instrument.
reconnect the software
(once disconnected)
to reconnect to an open plate document:
1. Select File > Close to close the plate document.
2. Click
(or select File
> Open).
3. In the Look In field, navigate to and select the plate
document of interest.
4. Click
,
Upon opening the plate document, the software
re-establishes the 7900HT instrument connection.
to reconnect to an new plate document, click
(or select File > New).
Upon creation of the plate document, the software
re-establishes the connection with the 7900HT instrument.
4-30
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Section 4.3 Automated Operation
Section 4.3 Automated Operation
In This Section
Pre-Run
Checklist
Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32
Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34
Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-39
After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44
The following tasks must be complete to run plates on the 7900HT instrument. See
the associated page number for details on each procedure.
Done
Check
See Page
A background run has been performed in the last week
7-16
A pure dye run has been performed in the last 6 months
7-20
The instrument tray does not contain a plate
4-44
IMPORTANT! The instrument tray must be empty to begin a run. If
the instrument tray contains a plate, you must eject and remove it.
The output stack does not contain plates.
Overview
–
The Applied Biosystems 7900HT Fast Real-Time PCR System features the capacity
for high-throughput unattended operation through the use of the Automation
Controller Software. The Automation Controller Software coordinates the function
of the 7900HT instrument, the fixed-position bar code reader, and the Zymark
Twister Microplate Handler to run batches of prepared plates with minimal user
intervention. The Automation Controller Software can run plates using plate
documents stored locally on the computer attached to the instrument, or across a
network on an SDS Enterprise Database.
Options for Automated Operation
As with the SDS Software, the Automation Controller Software requires the creation
of a plate document for each plate it runs. The Automation Controller Software can
run batches of plates using plate documents saved to the hard drive of the computer
attached to the instrument, or stored on an SDS Enterprise Database.
Follow the appropriate procedure for your system:
Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32
Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34
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4-31
Chapter 4 Operating the Instrument
Operating the Software with an SDS Enterprise Database
Overview
Running the Automation Controller Software with the SDS Enterprise Database is
the least complicated option for operating the Applied Biosystems 7900HT Fast
Real-Time PCR System. Plate documents saved to the SDS Enterprise Database are
automatically queued (set up for operation) for use on the Automation Controller
Software. Following plate document creation, plates can be prepared and loaded
immediately onto the Zymark Twister Microplate Handler for use.
Workflow
1. Start the Automation Controller Software and log in to the database (see
page 4-32).
2. If desired, set the study associations for the relative quantification and allelic
discrimination runs (see page 4-33).
3. Load prepared optical plates and Low Density Arrays onto the stacks of the
Zymark Twister Microplate Handler (see page 4-41).
4. Run the instrument (see page 4-43).
5. Analyze the run data.
– Allelic Discrimination Analysis (see page 5-5).
– Absolute Quantification Analysis (see page 6-5).
– Relative Quantification Analysis (see page 6-15).
– Dissociation Curve Analysis (see page 6-37).
Logging into the
Automation
Controller
Software
1. If running the SDS software, do one of the following:
• Select Instrument > Disconnect to discontinue communication between
the software and the instrument, or
• Select File > Exit to close the SDS software.
2. Start the Automation Controller Software
(select
> Programs > Applied Biosystems >
Automation Controller 2.2).
SDS 2.2.1 >
3. In the Login dialog box, enter your user name and password, then click
to log into the database.
4. Specify the Session-Study associations as described in “Associating Session and
Study Data” on page 4-33.
5. Prepare and load the plates onto the Plate Handler as explained in “Loading
Plates onto the Plate Handler” on page 4-41.
4-32
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Operating the Software with an SDS Enterprise Database
Associating
Session and
Study Data
User Access Requirement
If using the SDS Enterprise Database, all users can associate sessions with studies.
However, only users belonging to the Scientist or Administrator User Group can
create a new study.
About the Study Associations
The Automation Controller Software can automatically associate the data collected
from plate runs (called ‘sessions’) with a specific study. Attaching session data to
studies is optional and can be done later if necessary using the RQ or SNP Manager
Software. See “Database Design and Information Management” on page 1-27 for
more information about the use of studies.
IMPORTANT! After you have attached a session to a study, it cannot you cannot add it
to another study until you have unattached it using the RQ or SNP Manager Software.
To specify the Study associations:
1. In the Automation Controller Software dialog box, do one of the following:
To set the study association for:
• Relative Quantification runs – Click
in the Save Relative
Quantification to Study field (or select Enterprise > Select RQ Study).
• Allelic Discrimination runs – Click
in the Save Allelic
Discrimination to Study field (or select Enterprise > Select AD Study).
Click to set the study
association for relative
quantification runs
Click to set the study
association for allelic
discrimination runs
2. In the Select Study dialog box, either:
• Select an existing study, or
• Click
, and configure the Create New Study dialog box, then click
.
– Name – Enter a name for the study (up to 128 characters).
– Creator – (Not editable) Displays your user account name.
– Description – Enter a brief description of the study (up to 255 characters).
3. Click
.
4. Prepare and load the plates onto the Plate Handler as explained in “Loading
Plates onto the Plate Handler” on page 4-41.
IMPORTANT! The Save Analysis Results check box in the Database Options dialog
box must be selected for the Automation Controller Software to associate session
data (default is ON). When the option is selected, the software automatically
performs a primary analysis of each plate document it runs, and saves the results to
the database as an analysis session. To access the Save Analysis Results option by
selecting Enterprise > Database Options.
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4-33
Chapter 4 Operating the Instrument
Operating the Software without an SDS Enterprise Database
Overview
The first step in configuring the Applied Biosystems 7900HT Fast Real-Time PCR
System for automated operation is to add plate documents to the plate queue. The
plate queue is a list of plate document files that the Automation Controller Software
uses to identify and run associated plates during automated operation. By adding
plate documents to the queue, the plate documents automatically become available
for use with the Automation Accessory.
IMPORTANT! After you have added a plate document to the plate queue, the
software locks the file, preventing any changes from being made to it until you run or
remove the plate document from the queue.
The Applied Biosystems 7900HT Fast Real-Time PCR System has three options for
adding plate documents to the plate queue. Review the options listed below, then
choose the method that best suits your needs.
• Add a plate document to the plate queue from the SDS software. (page 4-35)
• Using the Template Batch utility, create batches of plate documents from a plate
document template and add them to the plate queue. (page 4-36)
• Add or remove individual or multiple plate documents to the plate queue using
the Automation Controller Software. (page 4-39)
Workflow
1. Add plate documents to the plate queue.
– Add a plate document to the plate queue using the SDS Software (see
page 4-35)
– Create and add plate documents to the plate queue using the Template
Batch Utility (see page 4-36)
2. Start and configure the Automation Controller Software and add or remove plate
documents from the plate queue as needed. (see page 4-39)
3. Load prepared optical plates and Low Density Arrays onto the stacks of the
Zymark Twister Microplate Handler. (see page 4-41)
4. Run the instrument. (see page 4-43)
5. Analyze the run data.
– Allelic Discrimination Analysis (see page 5-5)
– Absolute Quantification Analysis (see page 6-5)
– Relative Quantification Analysis (see page 6-15)
– Dissociation Curve Analysis (see page 6-37)
4-34
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Operating the Software without an SDS Enterprise Database
Adding a Plate Document to the Plate Queue (Using the SDS Software)
IMPORTANT! A plate document must contain a bar code before you can add it to the
plate queue. See page 3-25 for more information on configuring a plate document
with bar code information.
1. In the SDS software, select the Instrument tab.
2. In the Instrument tab, select the Queue tab.
3. In the Queue tab, click
.
Have you saved the plate document?
– Yes, then the software automatically saves the plate document.
– No, do the following:
a. In the Look in field of Save As dialog box, navigate to a directory where
the software can save the new file.
b. Select Files of type > SDS 7900HT Document (*.sds).
c. Click the File name field, then do one of the following:
– Enter a name for the plate document file.
– Enter or scan the bar code number of the plate into the field.
d. Click
. The software saves the plate document.
4. When prompted, click
to submit the document to the plate queue.
After you have added a plate document to the plate queue, the software locks the
file, preventing any changes from being made to it until the plate document is
run or removed from the queue.
Note: To release the plate document from the queue, start the Automation
Controller Software and remove the plate document from the queue, as
explained on page 4-40.
5. Click
.
6. Select File > Close. The SDS software closes the plate document.
7. Repeat the procedures in this chapter to create plates and add plates to the queue
as needed.
8. When you finish creating plate documents, run the queued plates as explained in
“Starting and Configuring the Automation Controller Software for Operation”
on page 4-39.
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4-35
Chapter 4 Operating the Instrument
Creating Plate Documents Using the Template Batch Utility
About the
Template Batch
Utility
The Template Batch utility allows you to quickly create multiple plate documents
from a single SDS 7900HT Template Document (*.sdt). The Template Batch utility
can be a useful timesaving device in situations where samples are run on plates with
identical assay configurations.
IMPORTANT! Plate documents created by the Template Batch utility do not contain
sample or plate information (Bar code or Comment settings). You must apply this
information to each plate document individually after the file is run.
Generating Plate
Documents from
a Template
Note: For more information on the elements of the Template Batch dialog box or to
view the procedures for importing or editing Plate IDs, click and see the Sequence
Detection Systems Software Online Help.
1. In the SDS software, open a plate document template from the:
Computer Hard Drive
a. Click
(or select File > Open).
b. Select File of type > SDS 7900HT Template Document (*.sdt).
c. In the Look in field, navigate to and select the plate document template.
d. Click
.
e. Go to step 2.
SDS Enterprise Database
a. Select File > Open Template from Database.
b. In the Template Names field of the Select Template dialog box, navigate
to and select the plate document template.
c. Click
d. Click
.
.
e. Save the plate document template to the computer hard drive as explained
on page 3-23.
f. Go to step 2.
The SDS software displays the plate document template.
2. Send the plate document template to the queue:
a. Select the Instrument tab.
b. Select the Queue tab.
3. Click
4-36
.
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Operating the Software without an SDS Enterprise Database
4. Configure the Template Batch dialog box with Plate IDs:
a. Click
.
b. In the Plate ID field of the New Plates dialog box, and scan the bar code of
the first plate in the batch using the hand-held bar code scanner.
LASER HAZARD. Exposure to direct or reflected laser
light can burn the retina and leave permanent blind spots. Never look into the
laser beam. Remove jewelry and anything else that can reflect the beam into
your eyes. Protect others from exposure to the beam.
IMPORTANT! If using the SDS Enterprise Database, the bar codes entered must
be unique and cannot be used by an existing plate document.
c. Repeat step b for every plate in the batch.
d. When finished, click
.
The plate bar codes appear inside the Plate ID field.
5. Select a destination directory to store the new plate documents:
a. Click
.
b. In the Look in field, navigate to and select the directory you want to use to
receive the new files.
c. Click
.
The Template Batch dialog box displays the selected destination directory in the
Plate Directory field.
Destination directory
6. Click
.
The software creates plate documents for all entries listed in the Plate ID list,
saves them to the destination directory, adds them to the plate queue, and
displays a message indicating the number of plate documents it created and sent
to the plate queue.
7. Click
8. Click
to close the message box.
to close the Template Batch dialog box.
9. Select File > Close to close the plate document template.
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4-37
Chapter 4 Operating the Instrument
10. Repeat the procedures in this Chapter 3, “Preparing a Run,” and Chapter 4,
“Operating the Instrument,” to create and add additional plates to the queue as
needed.
Note: After you have added a plate document to the plate queue, the software
locks the file preventing any changes from being made to it until the plate
document has been run or removed from the queue.
11. When finished creating and adding plate documents to the queue, run the queue
as explained in “Starting and Configuring the Automation Controller Software
for Operation” on page 4-39.
4-38
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Operating the Software without an SDS Enterprise Database
Starting and Configuring the Automation Controller Software for Operation
Starting and
Configuring the
Software
The Applied Biosystems 7900HT Fast Real-Time PCR System employs the
Automation Controller Software for automated operation of the 7900HT instrument.
The software coordinates the action of the 7900HT instrument, the bar code reader,
and the Plate Handler while acquiring and saving raw data during each run.
1. If running the SDS software, do one of the following:
• Select Instrument > Disconnect to discontinue communication between
the software and the instrument, or
• Select File > Exit to close the SDS software.
2. Start the Automation Controller Software
(select
> Programs > Applied Biosystems >
Automation Controller 2.2).
SDS 2.2.1 >
3. If using the SDS Enterprise Database, enter your user name and password in
the appropriate fields of the Automation Controller Log In dialog box, then
click
.
4. If using the SDS Enterprise Database, specify the Session-Study associations as
described in “Associating Session and Study Data” on page 4-33.
5. If running the Automation Controller Software using the plate queue, verify that
Plate Queue field contains plate documents for all plates you intend to run.
• To add a plate document to the plate queue, see “Adding Plates to the
Plate Queue” on page 4-40.
• To remove a plate document from the plate queue, see “Removing Plate
Documents from the Plate Queue” on page 4-40.
Plate Queue
(must contain plate
documents for all
plates to be run)
6. Prepare and load the plates onto the Plate Handler as explained on page 4-41.
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4-39
Chapter 4 Operating the Instrument
Adding Plates to
the Plate Queue
1. In the Automation Controller Software, select File > Add Plates.
2. In the Look in field of the Open dialog box, navigate to the directory containing
the file or files of interest.
3. While pressing and holding the Ctrl key, select the plate document file(s) to add
to the plate queue.
The software highlights selected files.
IMPORTANT! A plate document must contain a bar code before you can add it
to the plate queue. See page 3-25 for more information on configuring a plate
document with bar code information.
4. Click
.
The Automation Controller Software adds the plate document(s) to the Plate
Queue.
Note: After you have added a plate document to the plate queue, the software
locks the file preventing any changes from being made to it until the plate
document has been run or removed from the queue.
Removing Plate
Documents from
the Plate Queue
To remove plate documents from the plate queue:
To remove…
Then…
specific plate
documents from the
plate queue
1. While pressing and holding the Ctrl key, select the plate
document files to remove.
The software highlights selected files.
2. Select File
> Clear Selected Plate(s).
3. Click
.
The software removes the selected plate documents from the
plate queue.
all plate documents
from the plate queue
1. In the Automation Controller Software, select File >
Clear All Plates.
2. Click
.
The software removes all plate documents from the plate queue.
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Running Plates Using the Automation Controller Software
Running Plates Using the Automation Controller Software
User Access
Requirement
There is no access requirement. All users can run plates that have been saved to the
SDS Enterprise Database.
Loading Plates onto the Plate Handler
Guidelines
Observe the following guidelines when loading plates onto the Plate Handler:
• Before loading plates onto the Plate Handler, make sure that for each plate:
– the associated plate document has been added to the plate queue or is
contained in the SDS Enterprise Database
– the plate has been sealed using an optical adhesive cover.
• Load the plates into the Plate Handler stacks in any order. The software reads
the bar code of each plate before it is run and matches the plate document and
method with the plate. The Automation Controller Software can run batches of
up to 84 plates in a single session (21 plates/stack).
• Orient the plates inside the stacks so that well A1 ( ) of each plate corresponds
to the locations shown in Figure 4-11.
• Do not place plates in the output stack (X). The Plate Handler arm uses the
empty stack to store plates after it runs them.
• If loading Low Density Array(s) onto the Plate Handler:
– Load no more than 20 cards per stack.
– Pack stacks of cards tightly. (The Plate Handler may fail to detect a
misaligned card correctly when loading it.)
X
4
3
1
Zymark Twister Microplate
Handler (top view)
2
Bar code
Well A1
Figure 4-11
Plate Positions of the Zymark Twister Microplate Handler
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4-41
Chapter 4 Operating the Instrument
Loading Plates
1. Following the guidelines on page 4-41, load the sealed plates or cards into the
Plate Handler stacks.
2. In the Automation Controller Software, select the check boxes for the plate
stacks containing plates.
IMPORTANT! If you are not using stack 1 or the Restack option explained
below, remove all plates from stack 1 before starting the queue. Under these
settings, the Plate Handler will attempt to stack the run plates from stack 2 in the
stack 1 position. If stack 1 contains plates, these settings will cause the Plate
Handler to stop the run.
Plate stack check
boxes
3. If you want to retain the location of the plates in the stacks on the Plate Handler,
select the Restack when finished check box.
Restack when finished – Instructs the arm to replace a stack of used plates to
their original stack and in their original order after the stack has been run. If the
option is not selected, the arm will place each group of used plates inside the
next vacant stack in clockwise order beginning with the Output stack.
Note: Re-stacking plates adds significant operating time when running multiple
plates. Use the Restack when finished function only when necessary.
4. Begin the plate queue as explained on page 4-43.
4-42
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Running Plates Using the Automation Controller Software
Running the Instrument
Starting the
Plate Queue
After you have loaded all plate documents into the Plate Queue or saved them the
SDS Enterprise Database, and you have configured the Instrument Control options,
you can start the plate queue.
To begin the plate queue, click
from the Plate Queue tab.
The Automation Controller Software does the following:
1. The Zymark Twister Microplate Handler loads the first plate from Stack 1 and
places it into plate tray for the 7900HT instrument.
2. The fixed-position bar code reader scans the bar code for the plate.
3. The Automation Controller Software does one of the following:
• If linked to the SDS Enterprise Database, the software queries the database
for the plate document with the bar code of the associated plate. When
found, the software automatically downloads the plate document.
If the software does not find a plate document with a matching bar code in
the database, the software searches the contents of the plate queue as
explained below.
• If not linked to a database, the software searches the plate queue for the
plate document with the bar code of the associated plate. If found, the
Automation Controller Software loads the plate document information
from the file system for the run.
4. The 7900HT instrument loads the instrument tray and runs the plate according
to the parameters defined by the plate document file.
Note: Before starting a real-time run, the instrument may pause (up to
15 minutes) to heat the heated cover to the appropriate temperature.
If using the SDS Enterprise Database, the Automation Controller Software
automatically conducts a primary analysis of each completed run and saves the
results to the database. If you specified a study or studies as explained in
“Associating Session and Study Data” on page 4-33, then the software
automatically attaches the analyzed session data to the appropriate study.
The following options are available during and after the completion of the run:
To…
See Page
Monitor the progress of the run
4-44
Stop the run
4-44
IMPORTANT! If you must stop a run in-progress for any reason,
carefully read the instructions on page 4-28 before halting the run.
Open the instrument tray (after the run)
4-44
Analyze the run data after the run is complete
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4-43
Chapter 4 Operating the Instrument
Monitoring
Instrument
Progress
The Automation Controller Software displays the progress of the current run in the
Thermal Status tab. See page 4-27 for an explanation of the Thermal Status tab.
Stopping the
Instrument
To stop the plate queue, click
box at any time.
If you clicked
...
in the Automation Controller Software dialog
Then the instrument...
while the Plate Handler is
handling a plate
aborts the run and moves the Plate Handler to the home
position.
after a plate has been loaded
into the instrument, but
before the run has started.
aborts the current run, ejects the plate, and moves the
Plate Handler to the home position.
after the 7900HT instrument
has started a run.
aborts the current run, ejects the plate, and moves the
Plate Handler to the home position.
IMPORTANT! Stopping a run during thermal cycling can
affect the chemistry of the reactions inside the plate.
Before stopping a run, carefully read the guidelines on
page 4-28 to determine the best course of action.
Ejecting a Plate
To eject a plate following a halted run (or to open/close the instrument tray),
click
from the Automation Controller Software window.
After the Run
Analyzing the
Run Data
You can analyze the run data immediately following the completion of the run.
See the appropriate page for your application:
Section 5.1 Allelic Discrimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5
Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5
Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15
Section 6.3 Dissociation Curve Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-37
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Analyzing End-Point Data
In This Chapter
5
5
End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3
Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11
Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12
Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . 5-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18
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5-1
Chapter 5 Analyzing End-Point Data
End-Point Runs on the 7900HT Instrument
End-Point Runs
End-point is the term used to describe the category of sequence detection runs in
which the Applied Biosystems 7900HT Fast Real-Time PCR System is used to
measure the fluorescence of a biological sample after it has undergone thermal
cycling. Unlike real-time runs that can yield quantitative measurements, the focus of
end-point experiments is typically to produce a qualitative result. End-point analysis
is commonly used in combination with TaqMan® reagent-based chemistry to confirm
the presence or absence of specific target nucleic acid sequence in cells, tissues, or
fluid samples.
Currently, the SDS software supports one type of end-point analysis: Allelic
Discrimination.
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Notes for Database Users
Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when analyzing
Allelic Discrimination data as described in this chapter. This section describes the
types of actions you may need to perform depending on how your administrator has
configured the database. For more information on any of the features described
below, see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast
Real-Time PCR System Administrators Guide.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
When the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 5-1
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
When the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 5-2
Electronic Signature Verification Dialog Box Options
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5-3
Chapter 5 Analyzing End-Point Data
5-4
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Section 5.1 Allelic Discrimination
Section 5.1 Allelic Discrimination
In This Section
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11
Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12
Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . 5-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18
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5-5
Chapter 5 Analyzing End-Point Data
Overview
Allelic
Discrimination
on the 7900HT
Instrument
Employing the
5´ Nuclease
Assay for Allelic
Discrimination
The Applied Biosystems 7900HT Fast Real-Time PCR System supports allelic
discrimination using TaqMan® probes. Allelic discrimination is the process by which
two variants of a single nucleic acid sequence are detected in a prepared sample.
Allelic discrimination chemistry can be used for single-nucleotide polymorphism
(SNP) detection.
Allelic discrimination on the 7900HT instrument is made possible through the use of
the fluorogenic 5´ nuclease assay (see page D-2). During the PCR, the fluorogenic
probes anneal specifically to complementary sequences between the forward and
reverse primer sites on the template DNA. Then during extension, AmpliTaq Gold ®
DNA polymerase cleaves the probes hybridized to the matching allele sequence(s)
present in each sample. The cleavage of each matched probe separates the reporter dye
from the quencher dye, which results in increased fluorescence by the reporter. After
thermal cycling, the plate is run on the 7900HT instrument, which reads the
fluorescence generated during the PCR amplification. By quantifying and comparing
the fluorescent signals using the SDS software, it is possible to determine the allelic
content of each sample on the plate.
Mismatches between a probe and target reduce the efficiency of probe hybridization.
Furthermore, AmpliTaq Gold® DNA polymerase is more likely to displace the
mismatched probe than to cleave it, releasing the reporter dye. By running the extension
phase of the PCR at the optimal annealing temperature for the probes, the lower melting
temperatures (Tm) for mismatched probes minimizes their cleavage and consequently
their fluorescent contribution. Figure 5-3 illustrates results matches and mismatches
between target and probe sequences in TaqMan® PDARs for AD assays (Livak
et al., 1995; Livak et al., 1999).
Allele
X
F
V
Match
Probe-target sequence
Allele
Y
Mismatch
Probe-target sequence
mismatch (higher Tm)
V
VIC
F
FAM
Q
Quencher
V
F
Q
Q
Match
Probe-target sequence
Figure 5-3
Legend
Q
Q
AmpliTaq
Gold DNA
Polymerase
Mismatch
Probe-target sequence
mismatch (higher Tm)
GR1556
Target and Probe Sequence Interaction
Table 5-1 shows the correlation between fluorescence signals and sequences present
in the sample.
Table 5-1
5-6
Signal and Sequence Correlation
A substantial increase in…
Indicates…
VIC® dye fluorescence only
homozygosity for Allele X.
FAM™ dye fluorescence only
homozygosity for Allele Y.
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Overview
Table 5-1
Algorithmic
Manipulation of
Raw Allelic
Discrimination
Data
Signal and Sequence Correlation
A substantial increase in…
Indicates…
both fluorescent signals
heterozygosity.
The SDS software can analyze raw data immediately upon completion of an allelic
discrimination run. The term ‘raw data’ refers to the spectral data between 500 nm to
660 nm collected by the SDS software during the plate-read. During the analysis, the
software employs several mathematical algorithms to generate from the raw data a more
direct measure of the relationship between the spectra changes in the unknown samples.
The first mathematical algorithm involves the conversion of the raw data, expressed in
terms of Fluorescent Signal vs. Wavelength, to pure dye components using the extracted
pure dye standards. After the software identifies the dye components, it determines the
contribution of each dye in the raw data using the multicomponent algorithm.
About the
AutoCalling
System
After normalization, the software processes all samples associated with markers that are
configured for AutoCalling using the Applied Biosystems proprietary Maximum
Likelihood Algorithm. The algorithm conducts a cluster analysis of the data based on the
ratio of normalized reporter dye signal (for example, the ratio of FAM™ to VIC® dye).
The result of the analysis typically yields three major clusters corresponding to the three
genotypic constituents: Allele X homozygous, Allele Y homozygous, and heterozygous.
After clustering the data points, the software applies a series of probability tests to
further refine the clustered data and to identify outliers. For every sample, the software
calculates a final “quality value” that represents a measure of the likelihood that the
sample belongs to each cluster. The algorithm then applies the quality value for the
marker (set in the Analysis Settings dialog box) as a threshold for calling the associated
sample data. Sample-cluster associations that generate quality values greater than the
defined threshold are automatically assigned the call of the associated cluster.
Note: Any SNPs which depart substantially from expected Hardy-Weinberg
frequencies should be reviewed.
About the 2-Cluster Calling Feature
If only two clusters are present in a study, the AutoCalling algorithm uses the expected
genotype frequencies of the clusters to assign their genotypes. If a study contains two
large clusters and a single sample between the two clusters, the software considers the
intermediate sample as an outlier rather than a cluster, and treats the data as a two-cluster
study.
Note: The Maximum Likelihood Algorithm is designed to use No Template
Controls, and autocalling is most effective when NTCs are tasked.
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5-7
Chapter 5 Analyzing End-Point Data
Cluster Variations
The SDS software graphs the results of an allelic discrimination run on a scatterplot
contrasting reporter dye fluorescence (Allele X Rn versus Allele Y Rn). The software
represents each well of the 384-well plate as a datapoint on the plot. The clustering of
these datapoints can vary along the horizontal axis (Allele X), vertical axis
(Allele Y), or diagonal (Allele X/Allele Y). This variation is due to differences in the
extent of PCR amplification, which could result from differences in initial DNA
concentration.
The example below shows the variation in clustering due to differences in the extent
of PCR amplification.
Allele Y homozygotes
Allele X/Allele Y
heterozygotes
Outliers
Allele X homozygotes
No amplification
Figure 5-4
Common Datapoint Clusters
Genotypic Segregation of Datapoint Clusters
Figure 5-4 illustrates the concept of genotypic segregation of samples within the
allele plot. The plot contains four separate, distinct clusters that represent the No
Template Controls and the three possible genotypes (allele X homozygous, allele Y
homozygous, and heterozygous). Because of their homogenous genetic compliment,
homozygous samples exhibit increased fluorescence along one axis of the plot
(depending on the allele they contain). In contrast, heterozygous samples appear
within the center of the plot because they contain copies of both alleles, and therefore
exhibit increased fluorescence for both reporter dyes.
About Outliers
Samples that did not cluster tightly may:
• Contain rare sequence variations
• Contain sequence duplications
• Not contain a crucial reagent for amplification (the result of a pipetting error)
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Before You Begin
Before You Begin
Using the SDS
Software
Online Help
For specific instructions on any procedure described within this section, refer to the
Sequence Detection Systems Software Online Help. To get help at any time during the
procedure, click
located within the dialog box or window in which you are
working.
Examples in
This Section
The illustrations and screenshots that appear within this chapter were created from a
plate containing Pre-Developed TaqMan Assays and Reagents for Allelic
Discrimination run to screen 6 human genomic DNA samples (HD 1, 2, 3, 4, 7, 8) for
2 targets (CYP 2C9*2 and CYP 2C19*2). Each well of the plate contains 1 µL DNA,
TaqMan® 2✕ Universal PCR Master Mix, forward and reverse primers, and FAM and
VIC dye-labeled TaqMan probes.
Figure 5-5 illustrates the arrangement of the assays, unknown samples, and no
template control (NTC) wells on the plate.
HD-1 HD-1 HD-1 HD-1 HD-1 HD-1 HD-1 HD-1 HD-1 HD-1
HD-2 HD-2 HD-2 HD-2 HD-2 HD-2 HD-2 HD-2 HD-2 HD-2
HD-3 HD-3 HD-3 HD-3 HD-3 HD-3 HD-3 HD-3 HD-3 HD-3
NTC
HD-4 HD-4 HD-4 HD-4 HD-4 HD-4 HD-4 HD-4 HD-4 HD-4
HD-7 HD-7 HD-7 HD-7 HD-7 HD-7 HD-7 HD-7 HD-7 HD-7
HD-8 HD-8 HD-8 HD-8 HD-8 HD-8 HD-8 HD-8 HD-9 HD-8
GR2107
Not in Use
PDAR CYP 2C9*2
PDAR CYP 2C19*2
Figure 5-5
Configuration of the Samples and Detectors on the Example Plate
Note: The probes used in the example experiment were designed using the Primer
Express® Primer Design Software and by following the guidelines explained in
“Assay Development Guidelines” on page B-2.
IMPORTANT! The SDS software does not require that absolute quantification plates
contain positive controls.
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5-9
Chapter 5 Analyzing End-Point Data
Analysis Checklist
Where You Are in
the Procedure
1. Analyze the run data (see page 5-11).
2. View the results of the allelic discrimination run (see page 5-13).
3. Call allele types for each marker (see page 5-14).
4. Scrutinize the allele calls (see page 5-16).
5. Choose from the following post-analysis options (see page 5-18):
– Reanalyze the run data.
– Adjust the display settings for the plate document.
– Print elements of the plate document.
– Export the plate document results table or plots.
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Opening the Run Data
Opening the Run Data
User Access
Requirement
Opening the
Data from a
Completed Run
There is no access requirement. All users can open allelic discrimination data that
has been saved to the SDS Enterprise Database.
Opening a Plate Document File
1. Click
(or select File > Open).
2. In the Look in field, navigate to and select the plate document file.
3. Click
.
The SDS software displays the plate document file.
4. Configure the Analysis Settings for each marker as explained below.
Opening Un-Analyzed Data from the SDS Enterprise Database
1. Click
(or select File > Open Document from Database).
2. In the Plate Query dialog box, configure the Find Plates Matching These
Criteria table with parameters that correspond to the document of interest.
3. Click
.
Configure
queries here
Click to begin
the search
Documents that
meet the search
criteria appear
here
4. In the Search Results list, scroll to and select the session that you want to
analyze.
5. Click
.
6. Click
when finished.
7. Configure the Analysis Settings for each marker as explained below.
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5-11
Chapter 5 Analyzing End-Point Data
Analyzing an Allelic Discrimination Run
User Access
Requirement
About the
Analysis
There is no access requirement. All users can analyze allelic discrimination data.
The analysis of SNP (or genotyping) data involves the automatic or manual “calling”
of sample data for each marker. The “calls” are data labels assigned to individual
samples to reflect their genomic content. The SDS software can call sample data.
The SDS software can call sample data:
• Manually, using the toolbar and scatterplot.
• Automatically, using the “AutoCalling” system (see “About the AutoCalling
System” on page 5-7).
Configuring the
Analysis Settings
Before analyzing your data, you must configure the analysis settings for each marker
that you want to analyze using the AutoCalling algorithm. The analysis settings are
specific to each marker and must be set for each one individually.
IMPORTANT! You need to configure the analysis settings for a marker only if you
want to use the AutoCalling system to analyze it.
To configure the analysis settings:
1. Click
(or select Analysis > Analysis Settings).
2. Do one of the following:
To set the analysis settings for:
• A single marker, select a marker of interest from the Marker drop-down list.
• All markers, select All markers from the Marker drop-down list.
3. In the Analysis Settings dialog box:
a. Select the Auto caller enabled check box to activate the AutoCalling
system for the associated marker.
b. If you expect the analyzed data to consist of only two clusters, select the
2-Cluster Calling check box.
c. Enter a percentage value in the Quality field to apply as the quality interval
for AutoCalling samples.
4. Repeat steps 2 through 3 for the remaining markers on the plate document.
5. When finished, click
Analyzing the
Data
.
During the analysis, the software employs several algorithms to generate from the
raw data a more direct measure of the relationship between the spectral changes in
the samples.
To analyze the data:
1. Click
(or select Analysis > Analyze).
The SDS software analyzes the run data and displays the results in the Results tab.
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Calling and Scrutinizing Allelic Discrimination Data
Calling and Scrutinizing Allelic Discrimination Data
About the Allelic
Discrimination
View
The SDS software graphs the results of allelic discrimination runs on a scatterplot
contrasting reporter dye fluorescence. After signal normalization and
multicomponent analysis, the software graphs the normalized data from each well as
a single data point on the plot. Figure 5-6 illustrates the components of the Allelic
Discrimination plot.
Marker
drop-down list
Call
drop-down list
Toolbar
Scatterplot
Figure 5-6
Components of the Allelic Discrimination Plot
• Marker drop-down list – Determines the marker data that the software displays
within the plot.
• Call drop-down list – When a datapoint is selected, this menu allows you to
assign an allele call to the datapoint within the scatterplot.
• Toolbar – Contains the following tools for manipulating the plot.
Icon
Description
Selects individual data points by clicking or groups of datapoints by clicking
and dragging a box across a group of data points.
Selects groups of datapoints by encircling them with the tool.
Repositions the view within the plot by clicking and dragging the screen.
Zooms the plot by clicking the mouse button within the plot or by clicking
and dragging a section of the plot to view.
Zooms out on the plot by clicking the mouse button within the plot.
• Scatterplot – A scatterplot of data points from the run.
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Chapter 5 Analyzing End-Point Data
Manually Calling
Allele Types
1. Select the Results tab.
2. In the Allelic Discrimination Plot, zoom out until all crossmarks are visible in
the plot.
a. Click
(magnifying glass tool).
b. Click the plot to zoom out.
c. Click
(lasso tool).
d. Select all of the marks within the plot by clicking and dragging the pointer
across all datapoints in the plot.
The software outlines all selected wells within the grid view.
e. Examine the tray pane to confirm that all wells are selected. If not all wells
are selected, repeat steps a through d until all wells are visible on the plot.
Black border
(surrounding selected wells)
3. Select the sample cluster exhibiting amplification of the first probe.
Allele X
homozygotes
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Calling and Scrutinizing Allelic Discrimination Data
4. Select Call > Allele X.
Select Allele X
The software automatically labels the samples and wells with the Allele X call.
Allele X
homozygotes
5. Repeat steps 3 through 4 to apply calls to the rest of the samples within the plot.
Call
Symbol
Definition
Allele X
●
Allele Y
●
Both
●
Heterozygous (Alleles X and Y)
NTC
■
No Template Control
Undetermined
✕
Unknown (Unlabeled)
Homozygous for the allele displayed on the associated
axis of the Allelic Discrimination Plot.
Note: You can adjust the appearance of the allelic discrimination plot or the
data points it contains using the Display Settings dialog box. See the Sequence
Detection Systems Software Online Help for more information.
Allele Y homozygotes
Allele X/Allele Y
heterozygotes
Allele X homozygotes
No amplification
6. If evaluating for multiple markers, do the following:
a. In the Marker drop-down list, select a different marker.
b. Repeat steps 2 through 5 for the new marker.
c. Repeat steps a and b until the alleles for each marker have been called.
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Chapter 5 Analyzing End-Point Data
Scrutinizing the
Allele Calls
1. Verify the calls for the NTC, Allele X, and Allele Y controls.
a. In the plate grid, select the wells containing the No Template Control
samples.
The software highlights the datapoints within the allele plot.
b. Check that the datapoints cluster in the expected position on the plot.
c. If using positive controls, repeat steps a and b for the wells containing the
Allele X, and Allele Y controls.
Allele Y controls
should cluster here
Allele X controls
should cluster here
No Template
Controls should
cluster here
2. Designate samples that did not cluster tightly as Undetermined.
Samples that did not cluster tightly may:
• Contain rare sequence variations
• Contain sequence duplications
• Contain contaminants
• Be the result of errors in pipetting or sample preparation
Undetermined
samples
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Calling and Scrutinizing Allelic Discrimination Data
3. Screen for Unknown samples that failed to amplify:
a. In the Allelic Discrimination Plot, select the NTC cluster.
The SDS software highlights the datapoints within the allele plot and the
plate grid.
b. In the plate grid, check the wells containing Unknown samples for selected
wells that clustered with the NTCs.
Samples that clustered with the No Template Control wells may:
• Contain no DNA
• Contain PCR inhibitors
• Be homozygous for a sequence deletion
NTC cluster
(selected)
No Template
Control wells
Unknown samples
clustered with the NTCs
4. Retest any samples that did not cluster tightly or clustered with NTCs to
confirm the results.
5. If evaluating for multiple markers, do the following:
a. In the Marker drop-down list, select a different marker.
b. Repeat steps 1 through 4 for the new marker.
c. Repeat steps a and b until the calls for each marker have been verified.
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Chapter 5 Analyzing End-Point Data
After the Analysis
User Access
Requirement
Post-Analysis
Options
Changing the
Display Settings
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save the results of an analysis to the database.
The following options are available after the analysis:
Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-19
Before printing or exporting the analyzed data, you can reconfigure the appearance
of several elements of the plate document including the results table, plate grid, and
most plots (Allelic Discrimination, Raw Data, and Background plots).
1. Click
(or select View > Display Settings).
2. In the Display Settings dialog box, click
modifying the display settings.
Printing a Report
The SDS software can print a report of the analyzed data containing individual or
multiple elements of the plate document.
1. Click
(or select File > Print Report).
2. In the Print Report dialog box, click the
previewing, and printing the report.
Exporting Plate
Document Data
for further instructions on
for instructions on setting up,
Exporting Plate Document Data as a Tab-Delimited Text File
The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for
all or a select group of wells on a plate document. The exported files are compatible
with most spreadsheet applications and programs that can read tab-delimited text.
To export run data as a tab-delimited text file, choose one of the following for further
instructions:
• See “Exporting Plate Document Data” on page A-16.
• Click
within the table view.
Help button
Exporting Plots as Graphics
The SDS software can export most panes and plots of the plate document as JPEG
(Joint Photographic Experts Group) graphic files. The JPEG file format is
compatible with most word processing and spreadsheet applications and can be
incorporated directly into HTML documents for viewing by most web browser
software.
To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or click
within the plot of interest for further instructions.
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After the Analysis
Saving the
Results
The SDS software saves the results of the analysis differently depending on whether
the analysis is saved to the SDS Enterprise Database (see below) or to a plate
document file (see page 5-20).
Saving the Results to the SDS Enterprise Database
If you use the SDS Enterprise Database, you can save your analysis of the run data to
the database as an analysis session.
IMPORTANT! Observe the following when saving sessions to the database:
• If you modified the plate document information in any way (by removing wells
from use, or by changing the display settings), you must save the plate document
to the database for the changes to persist (select File > Save Plate Document to
Database).
• The SDS Enterprise Database allows two or more users to open and modify data
simultaneously. If another user has opened, analyzed, and saved the same data
that you have just analyzed, the software overwrites the analysis when you save
yours to the database.
• Analysis session results are directly linked to the configuration of the plate
document used to create the session. If, after you have saved your analysis
session to the database, you modify the linked plate document (by omitting
wells, altering sample names, etc.) your analysis session may be 'orphaned' (i.e.,
have no relationship to its attached plate document). Consequently, the results of
your analysis session may change if you reanalyze it because you have changed
the content of the associated plate document containing the raw data.
Applied Biosystems recommends the following guidelines for using analysis
sessions:
– Save the plate document as a new document any time you make changes to the
plate setup (omit wells, alter sample names, modify detectors or markers, etc.)
– Maintain only one saved analysis session per document
– Understand that only results sessions saved after the latest changes to a plate
setup are “guaranteed” to correspond to the linked plate document.
To save the results to the database:
1. Select File > Save Results to Database.
2. In the Description field of the Save Results to Database dialog box, enter a brief
description of the plate document (up to 255-characters).
Note: The software automatically populates the Session Name field with the
name of the associated plate document.
3. (Optional) In the Add to Study field, click
, then select an existing study,
or create a new study by clicking
, and doing the following:
a. In the Name field of the Configure the Create New Study dialog box, enter
a name for the study (up to 128 characters).
The Creator field is not editable and displays your user name.
b. In the Description field, enter a brief description of the study (up to
255 characters).
c. Click
.
d. Click
.
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Chapter 5 Analyzing End-Point Data
4. In the Save Results to Database dialog box, click
5. In the confirmation dialog box, click
.
.
Saving the SDS 7900HT Document
Although the software save any changes made to the appearance of a plate document
and to the analysis settings, it does not save the calls made during the analysis.
1. In the SDS software, select File > Save As.
2. In the Look in field of the Save As dialog box, navigate to and select a directory
for the software to receive the new file.
3. In the File name field, do one of the following:
• Enter a file name for the plate document file, or
• Enter or scan the bar code number for the plate into the field.
Note: The SDS software does not require that the file name match the bar code
of the corresponding plate.
4. Click
.
The software saves the plate document to the specified directory.
5-20
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Analyzing Real-Time Data
In This Chapter
6
6
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10
Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11
Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14
Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16
Essential Experimental Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20
Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22
Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . 6-23
Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23
Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26
Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35
Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Determining Tm Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44
Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45
Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46
Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
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Chapter 6 Analyzing Real-Time Data
Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when analyzing
data as described in this chapter. This section describes the types of actions you may
need to perform depending on how your administrator has configured the database.
For more information on any of the features described below, see the SDS Enterprise
Database for the Applied Biosystems 7900HT Fast Real-Time PCR System
Administrators Guide.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
When the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 6-1
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
When the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 6-2
6-2
Electronic Signature Verification Dialog Box Options
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Real-Time Runs on the 7900HT Instrument
Real-Time Runs on the 7900HT Instrument
Real-Time Runs
Real-time is the term used to describe the category of sequence detection runs in
which the Applied Biosystems 7900HT Fast Real-Time PCR System is used to
measure the fluorescence of a biological sample during thermal cycling. In contrast
to end-point runs, real-time experiments can be used to achieve both qualitative and
quantitative measurements. Real-time analysis can be used in combination with
either TaqMan® or SYBR® Green 1 double-stranded DNA binding dye reagents for a
variety of purposes including quantitative PCR and dissociation curve analysis.
Quantitative
RT-PCR
Quantitative RT-PCR is a method used to measure small quantities of ribonucleic
acid sequences isolated from biological samples. Typical biological samples include
cells, tissues, and fluids. During the RT step, reverse transcription of target RNA
produces corresponding complementary DNA (cDNA) sequences. During the
subsequent PCR, the initial concentration of target cDNA is quantified by amplifying
it to a detectable level. The two types of quantitative RT-PCR supported by the
7900HT instrument, absolute and relative, are described in detail below.
Absolute
Quantification
The objective of an absolute quantification experiment is to accurately determine the
absolute quantity of a single nucleic acid target sequence within an unknown sample.
The results of an absolute quantification experiment are reported in the same units of
measure as the standard used to make them.
Relative
Quantification
The objective of relative quantification is to determine the quantity of a single
nucleic acid target sequence within an unknown sample relative to the same sequence
within a calibrator sample. Relative quantification of target gene expression is
calculated from Applied Biosystems 7900HT Fast Real-Time PCR System data
using one of the following methods:
About the Standard Curve Method
The Standard Curve Method constructs a standard curve similar to that used in
absolute quantification experiments. However, because the goal of relative
quantification is to establish a fractional relationship, data produced by relative
quantification standards is used differently. For all experimental samples, target
quantity is determined from the standard curve and divided by the target quantity of a
calibrator sample. Because the unit from the standard curve drops out, only the
relative dilutions of the standard are necessary to determine relative quantities.
About the Comparative CT Method
The SDS software calculates relative levels of gene expression using the
Comparative CT Method of data analysis. The Comparative CT Method is similar to
the Standard Curve Method except that it uses arithmetic formulas to achieve the
same results for relative quantification. Instead of a standard curve, the Comparative
CT Method calculates relative gene expression using the equation:
Relative Quantity = 2–∆∆CT
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Chapter 6 Analyzing Real-Time Data
6-4
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Section 6.1 Absolute Quantification
Section 6.1 Absolute Quantification
In This Section
This section contains the following information:
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10
Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11
Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14
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Chapter 6 Analyzing Real-Time Data
Overview
About Absolute
Quantification
The Applied Biosystems 7900HT Fast Real-Time PCR System supports real-time
absolute quantification of nucleic acids using a standard curve method. The objective
of absolute quantification is to accurately determine the absolute quantity of a single
nucleic acid target sequence within an unknown sample. The results of an absolute
quantification experiment are reported in the same unit of measure as the standard
used to make them.
Employing the
5´ Nuclease
Assay
Absolute quantification on the 7900HT instrument is accomplished through the use
of the polymerase chain reaction and the fluorogenic 5´ nuclease assay (see
page D-2). During setup, standards diluted over several orders of magnitude and
unknown samples are loaded onto an optical plate containing master mix and
TaqMan reagents targeting a specific nucleic acid sequence. The plate is then loaded
into a 7900HT instrument which has been configured to perform a real-time run.
During the thermal cycling, the instrument records the emission resulting from the
cleavage of TaqMan® probes in the presence of the target sequence. After the run, the
SDS software processes the raw fluorescence data to produce threshold cycle (CT)
values for each sample (see page D-6). The software then computes a standard curve
from the CT values of the diluted standards and extrapolates absolute quantities for
the unknown samples based on their CT values (see below).
Note: See Appendix D, “Theory of Operation,” for more information on the
fluorogenic 5´ nuclease assay, real-time data collection, or the mathematical
transformations of sequence detection data.
Figure 6-3 illustrates a standard curve generated from a standard TaqMan® RNase P
Instrument Verification Plate. The arrangement of the samples and standards on the
plate are shown in “Examples in This Chapter” on page 6-8.
Standard Curve Plot
28
26
CT
Unknowns
24
22
20
1.0 E+2
1.0 E+3
1.0 E+4
1.0 E+5
Quantity
(LogN initial concentration)
Figure 6-3 Standard Curve Generated from a TaqMan RNase P Instrument
Verification Plate
6-6
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Overview
Algorithmic
Manipulation of
Raw Data
The SDS software can analyze raw data immediately after an absolute quantification
run is complete. The term raw data refers to the spectral data between 500 nm to
660 nm collected by the software during the real-time run. During the analysis, the
software automatically applies several mathematical transformations to the raw data
to generate a more direct measure of the relationship between the spectral changes in
the unknown samples.
Multicomponenting
The first mathematical transformation involves the conversion of the raw data,
expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components
using the extracted pure dye standards. At the same time, the software determines the
contribution of each dye in the raw data using the multicomponent algorithm.
Setting the Threshold and Calling CTs
After normalization, the baseline and threshold values must be set for the run
(see “Kinetic Analysis/ Quantitative PCR” on page D-4 for more information). The
results of the experiment can be visualized in the Standard Curve graph of the
Results tab. The graph consists of a scatterplot of standard and unknown samples
graphed on a linear-scale plot of Threshold Cycle (CT) versus quantity value.
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Chapter 6 Analyzing Real-Time Data
Before You Begin
Using the SDS
Software
Online Help
Examples in This
Chapter
For specific instructions on any procedure described within this section, refer to the
Sequence Detection Systems Software Online Help. To get help at any time during the
procedure, click
located within the dialog box or window in which you are
working.
The illustrations and screenshots that appear within this chapter were created for a
TaqMan® RNase P 384-Well Instrument Verification Plate (PN 4323306), an
experiment run during the installation of the 7900HT instrument to verify its
performance. The sealed plate is preloaded with the reagents necessary for the
detection and quantification of genomic copies of the human RNase P gene (a
single-copy gene encoding the RNA moiety of the RNase P enzyme). Each well
contains pre-loaded reaction mix (TaqMan® 2✕ Universal PCR Master Mix, RNase
P primers, and FAM™ dye-labeled probe) and template.
Population 2
10000
GR2107
Population 1
5000
NTC
STD 1250
STD 2500
STD 5000
STD 10000
STD 20000
Figure 6-4 illustrates the arrangement of standards and samples on the RNase P
Instrument Verification Plate. As shown below, the RNase P Instrument Verification
Plate consists of 5 columns of template standards (1250, 2500, 5000, 10,000, and
20,000 copies) and two replicate populations (5000 and 10,000 copies).
Figure 6-4
6-8
Sample Configuration of the Example Plate
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Analysis Checklist
Analysis Checklist
Workflow
Overview
1. Open the plate document (see page 6-10).
2. Configure the analysis settings for the run (see page 6-11).
3. Analyze the run data (see page 6-12).
4. For each detector that you configured to determine the baseline and threshold
values (see page 6-46):
– Automatically – Verify that the baseline and threshold values for the detector
were set correctly by the software.
– Manually – Set the baseline and threshold values for the detector
5. Visualize outliers and eliminate any outlying amplification from the run data
(see page 6-49).
6. View the results of the absolute quantification run (see page 6-14).
7. Choose from the following post-analysis options (see page 6-52):
– Reanalyze the run data.
– Adjust the display settings for the results table, plate grid, and plate
document plots.
– Print elements of the plate document.
– Export the plate document results table or plots.
– Save the analysis.
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Chapter 6 Analyzing Real-Time Data
Opening the Run Data
User Access
Requirement
Open a
Plate Document
There is no access requirement. All users can open absolute quantification data that
has been saved to the SDS Enterprise Database.
Opening a Plate Document File
1. Click
(or select File > Open).
2. In the Look in field, navigate to and select the plate document file.
3. Click
.
The SDS software displays the plate document file.
4. Configure the Analysis Settings for each marker as explained in “Configuring
the Analysis Settings” on page 6-11.
Opening a Plate Document from the SDS Enterprise Database
5. Click
(or select File > Open Document from Database).
6. In the Plate Query dialog box, configure the Find Plates Matching These
Criteria table with parameters that correspond to the document of interest.
7. Click
.
Configure
queries here
Click to begin
the search
Documents that
meet the search
criteria appear
here
8. In the Search Results list, scroll to and select the document that you want to
analyze.
9. Click
.
10. When the software has loaded the plate document, click
.
11. Configure the Analysis Settings for each marker as explained in “Configuring
the Analysis Settings” on page 6-11.
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Analyzing the Data
Analyzing the Data
User Access
Requirement
Configuring the
Analysis Settings
There is no access requirement. All users can analyze absolute quantification data
that has been saved to the SDS Enterprise Database.
Before analyzing the plate document data, you must configure the analysis settings
for each detector that you want to analyze. The analysis settings are detector-specific
and must be set for each one individually. For each detector, the SDS software can
calculate baseline and threshold values automatically using the AutoCT algorithm, or
manually using values you supply.
Note: You can analyze the run without configuring the analysis settings, however the
software will analyze the data using a default baseline of cycle 3 to 15 and a default
threshold value of 0.2.
1. Click
(or select Analysis menu > Analysis Settings).
2. In the Detector drop-down list, select a detector.
3. In the Analysis Settings dialog box, configure the method (automatic or manual)
that the software will use to determine the baseline and threshold values for the
selected detectors:
To determine the baseline and threshold values:
Automatically – Select the Automatic CT option button.
IMPORTANT! If you choose to use the AutoCT algorithm, verify that the
baseline and threshold were called correctly for each autocalled detector
following the analysis (use the procedure on page 6-46).
Note: See “Automatic Threshold and Baseline Determination” on page 6-18 for
more information on the AutoCT algorithm.
Manually – Do the following:
a. Select the Manual Ct option button.
b. In the Threshold field, enter a threshold value to apply to the selected
detector(s) or leave the field empty and set the threshold value manually as
explained on page 6-46.
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Chapter 6 Analyzing Real-Time Data
c. Do one of the following. Select the:
Automatic Baseline option button to have the software calculate a baseline
for the selected detector(s).
Manual Baseline option button, and enter Start and End cycle values to
apply to the selected detector(s).
Manual Baseline option button, and leave the Start and End cycles fields
empty. (You will need to set the values manually as explained on page 6-46.)
4. If necessary, repeat steps 2 through 3 for any additional detectors.
IMPORTANT! The threshold and baseline values set in step 3 on page 6-11 are
detector specific, and must be set for each individual detector used on in the
plate document.
5. Click
to accept the analysis settings and exit.
6. Analyze the data as explained below.
Analyzing the
Run
After you have configured the analysis settings, you can analyze the run data. During
the analysis, the software mathematically transforms the raw data to establish a
comparative relationship between the spectral changes in the passive reference dye
and those of the reporter dye. Based on that comparison, the software calculates a
cycle threshold (CT) for each reaction (standard and unknown). The software then
generates a standard curve for the run by plotting the standard samples on a graph of
CT versus initial copy number.
Note: See Appendix D, “Theory of Operation,” for a detailed description of the SDS
software mathematical transformation of real-time run data.
1. Click
(or select Analysis > Analyze).
The SDS software analyzes the run data and displays the results in the Results
tab.
2. Choose from the following:
• If using the AutoCT algorithm to set the baseline and threshold values for
the detectors on the plate, verify that the software has called the settings
correctly for each detector as described on page 6-46,
• If you choose to set the baseline and threshold values manually, set them
for each detector on the plate as explained on page 6-46.
3. If you choose to remove outliers manually, visualize and eliminate outliers from
the analyzed run data as explained on page 6-46.
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Viewing Results
Viewing Results
Viewing the
Standard Curve
The software displays the standard curve generated from the run data within the
Results tab of the plate document. The standard curve plot displays the unknown
samples on a graph of CT (threshold cycle) vs. Quantity (LogN). Figure 6-5
illustrates the components of the standard curve plot.
Help button
Detector drop-down list
Legend box
Hide Unknowns button
Standard Curve box
Standard curve plot
Figure 6-5
Components of the Standard Curve Plot
•
– Starts the Sequence Detection Systems Software Online Help.
• Detector menu – Toggles the data displayed within the plot based on detector
name.
• Legend box – Displays a symbol key for the datapoints appearing in the plot.
• Hide Unknowns button – Toggles the presence of data from unknown samples
in the plot.
• Standard Curve box – Contains the following statistical data describing the
standard curve.
– Slope – The slope of the standard curve. The slope of the standard curve is
useful for assessing the efficiency of the assay. At 100% efficiency, a reaction
should achieve a slope of −3.33 since every 10-fold difference in quantity
translates to a difference of 3.33 CTs.
– Y-Inter – The Y-axis intercept of the standard curve.
– R2 – The R square value for the standard curve that describes the correlation
between threshold cycles (CT) and the log of the quantity value for the
samples that comprise the standard curve plot. The calculation yields a value
between 1 and 0, where values closer to 1 indicate better correlation between
CT and the log of the quantity value.
Note: The software calculates the R square value by taking the square of the
Pearson Coefficient of Correlation (also known as the r value) calculated for the
data points that comprise the plot. The software calculates the R2 value only for
the standards that make up the curve.
• Standard curve plot – A scatterplot of datapoints from the absolute
quantification run.
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Chapter 6 Analyzing Real-Time Data
Viewing the
Analysis Table
Displays the results of the absolute quantification calculation in the results table of
the plate document. Figure 6-6 shows an example of the results table containing the
data from an RNase P Instrument Verification Plate.
Help button
Figure 6-6
Analysis Table of the Absolute Quantification Plate Document
The columns of the table are:
•
•
•
•
Position – The coordinate position of the well on the plate.
Sample – The sample name applied to the well.
Detector – The name of the detector assigned to the well.
Task – The task (NTC, Standard, or Unknown) assigned to the well.
Note: See page 3-14 for information on applying tasks to the plate document.
• CT – The threshold cycle generated by the well during the PCR.
Note: The software displays ‘Undetermined’ in the CT column when the
associated sample fails to produce a noticeable rise in fluorescent signal during
the entire PCR (the fluorescent signal produced by the well never crosses the
threshold defined for the associated detector).
• Quantity – For wells containing:
– Unknown samples, this column reports the extrapolated quantity value of the
sample in the same units of measure as the standard.
– Standard samples, this column displays the quantity assigned to the well.
Note: See page 3-15 for information on applying quantities to standard wells of
the plate document.
The following two columns contain data only if a well is run as part of a replicate group.
• Qty Mean – The arithmetic mean for the quantity values of the replicate group
associated with the well.
• Qty stddev – The standard deviation of the quantity values of the replicate
group associated with the well.
• Status – Indicates the analysis status of the sample data.
After the Analysis
User Access
Requirement
Post-Analysis
Options
6-14
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save the analyze data of an absolute quantification run.
Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Section 6.2 Relative Quantification
Section 6.2 Relative Quantification
In This Section
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16
Essential Experimental Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20
Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22
Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . 6-23
Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23
Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26
Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35
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Chapter 6 Analyzing Real-Time Data
Overview
Relative
Quantification on
the 7900HT
Instrument
The Applied Biosystems 7900HT Fast Real-Time PCR System supports real-time
relative quantification of nucleic acids using the Comparative CT method. The
Comparative CT Method uses the following arithmetic formula to achieve results for
relative quantification:
2 –∆∆CT
where:
∆∆CT =
The normalized signal level in a sample relative to the normalized signal level
in the corresponding calibrator sample.
Note: The derivation of the above equation is explained in ABI PRISM 7700 Sequence
Detection System User Bulletin #2: Relative Quantification of Gene Expression
(PN 4303859).
Note: The normalized signal levels discussed above refer to the signals generated by
the amplification of the target sequence in the unknown and calibrator samples which
is normalized to the signal generated by the amplification of the endogenous control.
Note: The SDS software does not support the Standard Curve Method of relative
quantification.
Employing the
5´ Nuclease
Assay
Relative quantification on the 7900HT instrument is accomplished through the use of
the polymerase chain reaction and the fluorogenic 5´ nuclease assay. After careful
selection of an endogenous control, probe and primer sets are designed for both the
target and control sequences. After the assays are verified, unknown samples are
loaded onto an optical plate containing master mix and 5′-nuclease assays targeting a
specific nucleic acid sequence. The reaction plate is then run on a 7900HT
instrument that is configured for real-time analysis.
During the run, the instrument records the emission resulting from the cleavage of
TaqMan® probes in the presence of the target sequence. After the run, the SDS
software processes the raw fluorescence data to produce threshold cycle (CT) values
for each sample. The SDS software computes relative quantities from the CT values
of the calibrator sample and the data from the unknown samples within the gene
expression profile.
Note: See Appendix D, “Theory of Operation,” for more information on the
fluorogenic 5´ nuclease assay, real-time data collection, or the mathematical
transformations of sequence detection data.
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Overview
Algorithmic Manipulation of Raw Data
The SDS software can analyze raw data immediately after a relative quantification
run is complete. The term “raw data” refers to the spectral data between 500 nm to
660 nm collected by the software during the real-time run. During the analysis, the
software automatically applies several mathematical transformations to the raw data
to generate a more direct measure of the relationship between the spectral changes in
the unknown samples.
Multicomponenting
The first mathematical transformation involves the conversion of the raw data,
expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components
using the extracted pure dye standards. At the same time, the software also
determines the contribution of each dye in the raw data using the multicomponent
algorithm.
Setting the
Threshold and
Calling Threshold
Cycles
After multicomponenting, the baseline and threshold values must be set for each
detector in the study. The software allows you to set the baseline and threshold values
manually (see page 6-49) or automatically (see page 6-19). The CT values computed
using the baseline and threshold data are the basis for generating gene expression
values.
Outlier Removal
For any PCR, experimental error may cause some wells to amplify insufficiently or
not at all. These wells typically produce CT values that differ significantly from the
average for the associated replicate wells. If included in the relative quantification
calculations, these outliers can potentially result in erroneous measurements. To
ensure precise relative quantification, replicate groups must be carefully scrutinized
for outlying wells before the analysis. The software provides options for removing
outliers automatically (see page 6-19) or manually (see page 6-49).
Generating Gene
Expression
Values
The final stage in the analysis is the computation of gene expression values from the
CT data. The mathematical process for deriving relative quantification values is
briefly described on page D-8. For detailed information on the derivation of the
∆∆CT equation, relative quantification data transformations, or other aspects of
5´ nuclease chemistry in relation to relative quantification, Applied Biosystems
recommends the following references:
• For information on performing relative quantification using
5´ nuclease chemistry on 7900HT instruments, see:
– ABI PRISM 7700 Sequence Detection System User Bulletin #2: Relative
Quantification Of Gene Expression (PN 4303859)
– RQ Manager Software User Guide(PN 4351670)
– The bibliography of this document for a list of technical papers and research.
• For information on the derivation of the ∆∆CT Equation, see:
– ABI PRISM 7700 Sequence Detection System User Bulletin #2: Relative
Quantification Of Gene Expression (PN 4303859)
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Chapter 6 Analyzing Real-Time Data
Automatic
Threshold and
Baseline
Determination
The SDS software features a proprietary AutoCT algorithm that can be used to
automatically generate baseline and threshold values for individual detectors. The
algorithm calculates baseline and threshold parameters for a detector based on the
assumption that the data exhibits the “typical” amplification curve shown in
Figure 6-7.
Plateau phase
Linear phase
Geometric phase
∆Rn
Background
Cycle
Figure 6-7
Baseline
Typical Amplification Curve
Experimental error (such as contamination, pipetting inaccuracy, and so on) can
produce amplification curves that deviate significantly from the typical data shown
above. The data from these irregularities can affect the AutoCT algorithm by causing
it to generate incorrect baseline and threshold parameters for the associated detector.
For this reason, Applied Biosystems strongly recommends reviewing all baseline and
threshold parameters determined by the AutoCT algorithm following analysis of the
study data.
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Overview
Automatic Outlier Removal
About Outlying
Replicate Wells
About the
Automatic Outlier
Removal
For any PCR, experimental error may cause some wells to amplify insufficiently or
not at all. These wells typically produce CT values that differ significantly from the
average for the associated replicate wells. If included in the calculations, the CT
values from these wells indicate an incorrect level of relative gene expression by
skewing the average for the replicate group.
The SDS software offers algorithmic identification and removal of outlying data for
studies consisting of replicate populations of three or more wells. The statistical
method used by the software is based on Grubbs outlier removal (also known as the
Maximum Normalized Residual Test), which permits the exclusion of a single outlier
in a population consisting of as few as three replicates.
The software applies the Grubbs tests at different stages in the ∆∆CT calculation,
depending on the type of endogenous control used in the study. For non-multiplex
studies, the software removes outliers from replicate groups immediately after
calculating threshold cycles (CT). For multiplex studies, the software applies the
algorithm following the ∆CT calculation. Also, if an apparent outlier is within 0.25
CTs of the mean for the associated replicate group, the software does not remove it.
Note: The outlier removal feature is optional and can be activated from the Analysis
Settings dialog box (see page 6-29). Outliers can also be identified and eliminated
manually as explained on page 6-49.
In addition to the Grubbs tests, the algorithm used by the SDS software features
additional rules for dealing with SDS data. Unless the majority of samples in a
replicate population are at maximum cycle, the algorithm ignores max cycle wells
(wells with CTs equal to or greater than the Maximum cycle value).
Regarding
Amplification
Beyond
Reproducible
Limits
The experimental reproducibility of CT values for relative quantification assays
decreases as the starting copy number approaches extremely low levels (less than
100 copies). Therefore, PCR targets with low starting copy number and subsequent
high CT values may have significantly diminished statistical reproducibility. To avoid
problems with reproducibility for low-expression targets, the limits of reproducibility
must be determined experimentally for each relative quantification assay (all targets
and the endogenous control).
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Chapter 6 Analyzing Real-Time Data
Essential Experimental Components
Endogenous
Control
All relative quantification experiments require data from a second probe and primer
set that amplifies an endogenous control sequence. Endogenous control targets are
typically constitutive RNA or DNA sequences that are present at a statistically
consistent level in all experimental samples. By using the endogenous control as an
active reference, the data from the amplification of a messenger RNA (mRNA) target
can be normalized for differences in the amount of total RNA added to each reaction.
Examples of common endogenous controls are: GAPDH, 18S rRNA, and β-Actin.
The endogenous control can be amplified independently of the target sequences in
separate wells on the reaction plate (as singleplex or non-multiplex reactions), or in
the same well (as multiplex reactions).
IMPORTANT! For non-multiplex experiments, the reactions amplifying the
endogenous control must be located on the same plate as the target assays.
IMPORTANT! To generate a standard deviation for the relative quantity value of a
target, each plate must contain usable data from at least two replicates of the target
and endogenous control.
Calibrator
Sample
All relative quantification experiments require data from a calibrator sample. During
analysis, the SDS software calculates gene expression levels in samples relative to
the level of expression in a calibrator sample. Thus, the calibrator plays an integral
part in the calculation because it is used as the basis for the comparative results.
Examples of possible calibrator samples include:
• A zero time-point sample in a time-course experiment
• An untreated sample (versus treated samples)
• A resting sample (versus activated samples)
Note: The SDS software combines the data from replicate calibrator wells at the ∆CT
level of the relative quantification calculation (whether the replicates are present on a
single or multiple plates).
Replicate Wells
For relative quantification studies, Applied Biosystems recommends the use of three
or more replicate reactions per sample to ensure statistical confidence.
Replicates allow you to:
• Preserve Data – If an amplification fails in one well, replicate wells can
potentially provide data. This point is especially true in the case of endogenous
controls which, upon failure, may invalidate the results for the entire plate.
• Remove Outliers – The use of replicate populations provide the opportunity for
the visualization and removal of outliers.
• Ensure Statistical Reproducibility – In general, the use of replicates ensures a
greater degree of experimental reproducibility by providing a means to identify
and refute anomalous data caused by experimental error (such as contamination,
pipetting errors, and so on).
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Getting Started
Getting Started
Using the SDS
Software
Online Help
For specific instructions on any procedure described within this section, refer to the
Sequence Detection Systems Software Online Help. To get help at any time during the
procedure, click
located within the dialog box or window in which you are
working.
Examples in This
Chapter
The screenshots that appear within this chapter were created using a series of
TaqMan® Cytokine Gene Expression Plates (PN 4304671), a research tool for
real-time, in vitro quantitative evaluation of human cytokine gene expression. The
plate detects the expression of 12 cytokine target sequences and an endogenous
control in complementary DNA (cDNA) samples. The TaqMan Cytokine Gene
Expression Plate I consists of a MicroAmp® Optical 96-Well Reaction Plate arranged
into 24 replicate groups, each containing TaqMan probes and primers for the assay of
one human cytokine mRNA and an endogenous control. Figure 6-8 illustrates the
assay configurations for the plate.
The study used to produce the screenshots for this section consisted of four plates of
a time-course experiment run to evaluate the expression of 24 target cytokine mRNA
over a 36-hour period.
36 hr
IL-3
IL-4
IL-5
IL-6
IL-7
IL-8
IL-10
IL-12p35
IL-12p40
IL-13
IL-15
IL-17
IL-18
G-CSF
GM-CSF
M-CSF
IFNγ
LTβ
TGFβ
TNFα
TNFΒ
Plate 1
GR2108
IL-1β
24 hr
Plate 2
GR2108
IL-1α
12 hr
Plate 3
GR2108
IL-2
Figure 6-8
Plate 4
GR2108
GR2108
0 hr
Sample Configuration of the Example Plates
Note: For more information on the TaqMan Cytokine Gene Expression Plate I, see
the TaqMan Cytokine Gene Expression Plate I Protocol (PN 4306744).
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Chapter 6 Analyzing Real-Time Data
Analysis Checklist
Workflow
Overview
1. Create an SDS 7900HT Multiple Plate Document (see page 6-23).
2. Add relative quantification plates for the analysis (see page 6-24).
3. Configure the analysis settings for the run (see page 6-26).
4. Analyze the run data (see page 6-30).
5. For each detector that you configured to determine the baseline and threshold
values (see page 6-46):
– Automatically – Verify that the baseline and threshold values for the detector
were set correctly by the software.
– Manually – Set the baseline and threshold values for the detector.
6. For each detector that you configured for manual outlier removal, visualize outliers
and eliminate any outlying amplification from the run data (see page 6-49).
7. View the results of the relative quantification run (see page 6-31).
8. Choose from the following post-analysis settings (see page 6-35):
– Reanalyze the run data.
– Adjust the display settings for the results table, plate grid, and plate
document plots.
– Print elements of the plate document.
– Export the plate document results table or plots.
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Options for Analyzing Relative Quantification Data
Options for Analyzing Relative Quantification Data
Applied Biosystems provides two ways to analyze data collected for relative
quantification experiments.
SDS Multiple
Plate Documents
(for Small-Scale
Analyses)
In the case of relative quantification, the SDS software uses a different kind of plate
document, called an SDS 7900HT Multiple Plate Document (*.sdm), to analyze data
from small relative quantification studies consisting of less than 10 plates. Unlike
file-based plate documents, multiple plate documents save the settings and results of
an analysis. Regardless of whether you are analyzing data from a single plate, or
from multiple plates in a series, you must create a multiple plate document to
conduct the analysis.
Note: The SDS software does allow you to open individual relative quantification
plate documents, however they cannot be used individually to generate gene
expression values.
RQ Manager
Software
(for Medium to
Large-Scale
Analyses)
The RQ Manager Software is a stand-alone application used to conduct medium- to
large-scale analyses of relative quantification (gene expression) data stored on an
SDS Enterprise Database. The software can conduct comparative analyses of over
97 detectors (96 target genes and 1 endogenous control) across 200 plates, or
approximately 153,600 multiplexed reactions run on an Applied Biosystems 7900HT
Fast Real-Time PCR System instrument. See the RQ Manager Software User Guide
(PN 4351670) for more information.
Creating the Study
Creating a
Multiple Plate
Document
To conduct a comparative analysis of a series of relative quantification plates in a
study, you must first create a Relative Quantification Multiple Plate Document.
1. In the SDS software, click
(or select File > New).
2. Configure the New Document dialog box with the following settings:
• Assay – Relative Quantification (∆∆CT) Study
• Container – the plate formats used to run the samples in the study.
IMPORTANT! Relative quantification studies cannot compare data from runs
performed using different plate formats (for example, samples run in a 96-well
plate cannot be compared to those run in a 384-well plate).
3. Click
attributes.
. The software displays a new plate document with the appropriate
4. Add the relative quantification plate documents to the study as explained in
“Adding Plate Documents to the Study” on page 6-24.
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Chapter 6 Analyzing Real-Time Data
Adding Plate
Documents to
the Study
To conduct a comparative analysis of a series of relative quantification plates in a
study, you must first add the plates to a Relative Quantification Multiple Plate
Document.
Plate documents added to a study must:
• Be of the same consumable format (for example, you cannot compare samples
run on 384-well plates to those run on 96-well plates)
• Have been run using the same method (identical thermal cycler conditions,
sample volume, and data collection options)
• Contain sample names for all In Use wells. (If a plate document does not
contain samples, you can apply sample names to it using the SDS software.)
IMPORTANT! Relative Quantification Multiple Plate Documents exclude from the
analysis all wells of plates that do not have a sample name applied to them.
IMPORTANT! Relative Quantification Multiple Plate Documents cannot compare
data from runs performed using different plate formats (for example, samples run in
a 96-well plate cannot be compared to those run in a 384-well plate).
To add plates to the Relative Quantification Multiple Plate Document:
1. In the multiple plate document, click
.
2. In the Add dialog box, do one of the following:
If you know where your plate documents are located:
a. Select the File tab.
b. Navigate to the directory containing the relative quantification plate
document files of interest.
c. Press and hold the Ctrl key, select the files of interest, then
click
.
Added plate
document files
Selected plate
document files
If you do not know where your plate documents are located, or want to search
for specific plate documents:
a. Select the Search tab.
b. In the Search in field, enter a directory address to search or click and select
a location (directory or hard drive) to search.
c. If you want the SDS software to search all subdirectories in the chosen
location, select Include Subdirectories.
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Creating the Study
d. In the Plate Query dialog box, configure the Find Plates Matching These
Criteria table with parameters that correspond to the documents of interest.
e. Click
.
f. In the Search Results list, press and hold the Ctrl key, select the files of
interest, then click
.
Plate documents
added to the
study
Query designed to find
plate documents with file
names containing “RQ“
Plate documents
found by the software
Note: See “Using the Search Tool” on page A-20 for more information on
searching for plate documents.
3. Repeat step 2 to add additional files to the study (up to 10 plates).
Note: You can remove plates from the study by selecting the file from the File
list field, and then clicking
.
4. When you finish adding files to the study, click
.
5. Configure the analysis settings for the study as explained in “Configuring the
Analysis Settings” on page 6-26.
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Chapter 6 Analyzing Real-Time Data
Analyzing the Study
User Access
Requirement
Configuring the
Analysis Settings
There is no access requirement. All users can analyze absolute quantification data
that has been saved to the SDS Enterprise Database.
Before analyzing the data for your study, you must specify the analysis settings for
each detector that you want to analyze. The settings are detector specific and must be
set for each detector individually. The following sections briefly discuss the analysis
settings provided by the software in the Analysis Settings dialog box:
• About the Analysis Settings (see below)
• Specifying the Analysis Settings (see page 6-29)
Note: You can analyze the run without configuring the analysis settings. However,
the software analyzes the data using the baseline of cycles 3 to 15 and a default
threshold value of 0.2.
About the Analysis Settings
CT Analysis for
Selected Detector
Settings
The settings of this group box control the method that the SDS software uses to
calculate the CTs of the sample replicate groups associated with the selected detector
in the Detector drop-down list. As shown in Figure 6-9, the Ct Analysis settings
provide both automatic and manual methods for determining the baseline and
threshold values.
Automatic Ct
option button
Manual Ct
option button
Figure 6-9
CT Analysis for Selected Detectors Settings
• Automatic Ct – Instructs the software to calculate the optimal baseline range
and threshold values for the selected detectors using the AutoCT algorithm
(see page 6-18).
IMPORTANT! If you choose to use the AutoCT algorithm, Applied Biosystems
recommends that you verify that the baseline and threshold values are set
correctly for each detector configured for AutoCalling (see page 6-46).
6-26
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Analyzing the Study
• Manual Ct – Instructs the software to use the baseline and threshold values
specified in the Analysis Settings dialog box to calculate CTs for the selected
detectors.
The software provides two methods for manually setting the baseline and
threshold values for the detector:
– If you know the values that you want to use, enter them into the appropriate
fields of the Analysis Settings dialog box.
– If you want to set the baseline and threshold using the controls of the
Amplification Plot tab, leave the fields empty and set the values later as
described on page 6-46.
Replicate and
Outlier Removal
for Study Settings
The settings of this group box determine how the software identifies and removes
outlier data for all detectors in the study.
Automatic Outlier
Removal check box
Omit replicates whose ∆CT <=…
check box
Figure 6-10
Replicate and Outlier Removal for Study Settings
• Automatic Outlier Removal – Instructs the software to automatically remove
the data of outlying replicate wells using the outlier removal algorithm. See
“Automatic Outlier Removal” on page 6-19 for an explanation of the algorithm.
• Omit replicates whose ∆CT <=… – Instructs the software to remove from the
analysis, the data of any sample well whose ∆CT is less than the number of
cycles specified in the associated field. This option is only available for
multiplex experiments where similar target and endogenous control CTs may
indicate that competition of the assays is affecting the amplification.
Note: You can also identify and eliminate outliers manually, as explained in
“Eliminating Outlying Amplification” on page 6-49.
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Chapter 6 Analyzing Real-Time Data
Gene Expression
Settings for Study
Settings
The settings in this group box determine how the software handles the endogenous
control data and RQ Min/Max confidence values for your study. These settings are
crucial to the analysis and are set once for the study.
Calibrator Sample drop-down list
Endogenous Control Detector
drop-down list
Control Type option buttons
RQ Min/Max Confidence drop-down list
Figure 6-11
Gene Expression Settings for Study Settings
• Calibrator Sample – Displays the name of the sample that the software uses as
the calibrator for the study.
• Endogenous Control Detector – Specifies the name of the detector that the
software will use as the endogenous control for the study.
• Control Type – The Multiplexed/Non-Multiplexed option buttons specify how
the software will use the data from the wells labeled with the endogenous
control data.
• RQ Min/Max Confidence – Specifies the confidence value that the software
will use to calculate the standard error of the mean expression level (RQMax
and RQMin values) for the samples in the study.
• Export Individual ∆CTs – Instructs the software to export the ∆CT values of
individual samples in the analysis instead of the average ∆CT values for the
replicate group.
6-28
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Analyzing the Study
Specifying the Analysis Settings
Specifying the
Settings for the
Analysis
1. Click
(or select Analysis > Analysis Settings).
2. In the Detector drop-down list, select:
• detector name – To set the Analysis Settings for a specific detector.
• All Detectors – To set the Analysis Settings for a all detectors
simultaneously.
3. In the Analysis Settings dialog box, configure the method (automatic or manual)
that the software will use to determine the baseline and threshold values for the
selected detectors:
To determine the baseline and threshold values:
Automatically
Select the Automatic Ct option button. (See page 6-18 for information on the
Automatic Ct algorithm.)
If you choose to use the Automatic CT algorithm, verify that the baseline and
threshold were called correctly for each detector configured for AutoCalling
(following the procedure on page 6-46).
Manually
Do the following:
a. Select the Manual Ct option button.
b. In the Threshold field, enter a threshold value to apply to the selected
detector(s) or leave the field empty and set the threshold value manually as
explained on page 6-46.
c. Select one of the following:
• Automatic Baseline, to have the software calculate a baseline for the
selected detector(s).
• Manual Baseline, then enter Start and End cycle values to apply to the
selected detector(s).
• Manual Baseline, and leave the Start and End cycles fields empty. (You
will need to set the values manually as explained on page 6-46.)
4. If necessary, repeat steps 2 through 3 for any additional detectors.
IMPORTANT! The threshold and baseline values set in step 3 are detector specific,
and must be set for each individual detector used on in the plate document.
5. If evaluating groups of two or more replicates per sample, select or deselect the
Automatic Outlier Removal option.
6. If desired, select the Omit Samples Whose ∆CT <= check box, then enter a
value in the field. See “Replicate and Outlier Removal for Study Settings” on
page 6-27 for more information.
7. In the Calibrator Sample drop-down list, select the sample that you want to use
as the calibrator for your study.
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Chapter 6 Analyzing Real-Time Data
8. In the Endogenous Control Detector drop-down list, select the detector
representing the endogenous control for your study.
IMPORTANT! The detector used as the endogenous control must be present in
all sessions attached to the study.
9. Select the option button (Multiplexed or Non-Multiplexed) to indicate the type
of endogenous control used.
Note: The Multiplexed or Non-Multiplexed option buttons are active only if the
sessions loaded for analysis contain both multiplex and non-multiplex reactions.
10. In the RQ Min\Max Confidence drop-down list, select the confidence value
you want the software to use when generating error bars for the gene expression
plot.
11. If desired, select the Export Individual ∆CT option button to instruct the SDS
software to display the ∆CT values for individual samples belonging to replicate
groups. If the option is not selected, the software displays the average ∆CT for
the replicate group.
12. Click
to accept the analysis settings and exit.
13. Analyze the data as explained in “Analyzing the Data” on page 6-30.
Analyzing the Study Data
Analyzing
the Data
During the analysis, the software mathematically transforms the raw data to establish
a comparative relationship between the spectra changes in the passive reference dye
and those of the reporter dye. Based on that comparison, the software calculates a
cycle threshold (CT) for each reaction (endogenous control and unknown).
Note: See Appendix D, “Theory of Operation,” for a detailed description of the SDS
software mathematical transformation of real-time run data.
1. Click
(or select Analysis > Analyze).
The SDS software analyzes the run data and displays the results in the
Results tab.
2. Choose from the following:
• If you choose to set the baseline and threshold values manually, set them
for each detector on the plate as explained on page 6-46.
• If using the AutoCT algorithm to set the baseline and threshold values for
the detectors on the plate, verify that the software has called the settings
correctly for each detector by following the procedure for manually setting
the baseline and threshold on page 6-46 without adjusting the settings.
3. If you choose to remove outliers manually, visualize and eliminate outliers from
the analyzed run data as explained on page 6-46.
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Viewing Results
Viewing Results
Displaying the
Analyzed Data
About the Gene
Expression Plot
The Gene Expression plot displays the data of the selected target detector(s) in the
Detectors list. The SDS software allows you to limit the detector and plate data
displayed in the Gene Expression plot through the Plates and Targets tables of the
Plate List Panel. The software displays data from the targets and plates in the lists
whose check boxes are selected.
The SDS software displays the graphic equivalent of the results of the relative
quantification (∆∆CT) calculations in the Gene Expression plot. The plot is in the
upper division of the multiple plate document. Figure 6-12 illustrates the important
components of the plot.
Error bar (see below)
Arrow (see page 6-31)
Legend
Plot
Detectors
Figure 6-12
Components of the Gene Expression Plot
• X–Axis – Lists all the targets involved in the analysis. Within each target group,
the software displays the relative quantity of the target in each sample.
• Y–Axis – The results of the relative quantification calculations are grouped by
target sequence, and the relative quantities are graphed on a logarithmic scale.
The quantities are shown relative to the expression level in the calibrator sample,
where each increment corresponds to a 10-fold difference in gene expression.
About the
Expression
Levels
The SDS software displays the results of the relative quantification calculations on a
logarithmic scale where each increment corresponds to a 10-fold difference in gene
expression. The software displays the expression level for each selected detector
relative to the expression level in the calibrator sample (shown in the Calibrator
drop-down list).
Note: Because the calibrator is compared to itself, the expression level for the calibrator
always appears as 1 (1E+00).
Table 6-1 on page 6-32 summarizes the circumstances in which the SDS software
displays gene expression levels for sample replicate groups. The shaded rows of the
table indicate the combinations of data for which the software cannot calculate
accurate expression levels.
Note: The expression minimum ( ↑) and maximum ( ↓ ) symbols are described in
detail on the page 6-33.
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6-31
Chapter 6 Analyzing Real-Time Data
Table 6-1
Summary of Expression Levels
Sample ∆CTs
Detector
Example (for a 40-cycle experiment)
Calib ∆CT
Test ∆CT
25
32
<
33
35
b
Calibrator
Test
Target
<a
<
Endo
<
b
Then expression
level is…
Displayed
Target
Max
<
A minimum
—
34
Endo
<
<
(bar displays ↑)
35
30
Target
<
Max
A maximum
33
—
Endo
<
<
(bar displays ↓ )
33
33
Target
Max
Max
Undetermined
—
—
Endo
Max
Max
(not displayed)
—
—
Target
<
Max
Undetermined
30
—
Endo
Max
Max
(not displayed)
—
—
Target
Max
<
Undetermined
—
30
Endo
Max
Max
(not displayed)
—
—
Target
Max
Max
Undetermined
—
—
Endo
Max
<
(not displayed)
—
30
Target
Max
Max
Undetermined
—
—
Endo
<
Max
(not displayed)
30
—
Target
<
<
Undetermined
30
20
Endo
Max
Max
(not displayed)
—
—
Target
<
Max
Undetermined
35
—
Endo
Max
<
(not displayed)
—
33
Target
<
Max
Undetermined
30
—
Endo
<
Max
(not displayed)
20
—
Target
Max
<
Undetermined
—
30
Endo
Max
<
(not displayed)
—
20
Target
Max
Max
Undetermined
—
—
Endo
<
<
(not displayed)
20
20
Target
Max
<
Undetermined
—
35
Endo
<
Max
(not displayed)
33
—
Target
<
<
Undetermined
30
33
Endo
<
Max
(not displayed)
35
—
Target
<
<
Undetermined
33
30
Endo
Max
<
(not displayed)
—
35
Result
• RQ is displayed
• Bar is displayed
• RQ is displayed
• Bar displays ↑
• RQ is displayed
• Bar displays ↓
• RQ value is not
calculated
• Bar is not displayed
a< : Indicates that the sample CT is less than the maximum PCR cycle of the run (a valid CT).
bMax (or —): Indicates that the amplification curves for the associated sample replicate group never crossed the threshold
during the duration of the run (‘undetermined’ values).
6-32
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Viewing Results
About Down
Arrows
Down Arrow
The software displays a down arrow to indicate that a level of expression represents
the maximum target gene expression level. The software displays the down arrow on
a sample bar when:
• The calibrator sample for the detector group yields valid target and endogenous
control CTs (CTs less than the maximum cycle), and
• A test sample yields an undetermined target CT (and a valid endogenous
control).
At the levels above, the test sample does not contain enough target cDNA for an
accurate comparison (with the calibrator sample). Therefore, the stated expression
level is the maximum possible value for the associated test sample.
About Up Arrows
Up Arrow
The software displays an up arrow to indicate that a level of expression represents the
minimum target gene expression level. The software displays the up arrow on all
associated sample bars when:
• The target sample for the detector group yields valid target and endogenous
control CTs (CTs less than the maximum cycle), and
• A calibrator sample yields an undetermined target CT (and a valid endogenous
control).
At the levels above, the calibrator sample does not contain enough target cDNA for
an accurate comparison with the test sample. Therefore, the stated expression level is
the minimum possible value for the sample.
About the
Error Bars
The software displays error bars for each column in the plot if the associated
expression level was calculated from a group of two or more replicates. The error bars
display the calculated maximum (RQMax) and minimum (RQMin) expression levels
that represent standard error of the mean expression level (RQ value). Collectively, the
upper and lower limits define the region of expression within which the true expression
level value is likely to fall. The software calculates the error bars based on the (RQMin
and RQMax) confidence interval setting in the Analysis Settings dialog box.
Note: Since standard deviation can only be calculated from two or more replicates,
the SDS software does not display error bars, RQMin and RQMax values, or standard
deviation (if either the target or the endogenous control has only a single sample).
Note: Error bars are a representation of the likelihood of a random sample meeting
acceptance criteria based on standard of population, degrees of freedom, and RQ
Min/Max confidence values. The software does not display error bars for the
calibrator sample because it is compared to itself.
Undetermined
Samples and
Outlier Removal
The software declares sample as undetermined if the amplification curves for the
associated replicates in the group do not cross the threshold for the detector during
the duration of the run.
• If outlier removal is not turned on, then the software ignores any undetermined
wells and displays the CT of the remaining wells as the average CT.
• If outlier removal is turned on, then voting occurs. If the majority of the wells in
the sample replicate group are undetermined, then the software displays the
average CT as undetermined. If half or more of the wells have valid CT values,
then the software ignores the undetermined wells, removes outliers from the
remaining wells (if applicable), and averages the resulting data.
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6-33
Chapter 6 Analyzing Real-Time Data
Viewing Relative
Quantities in the
Analysis Table
The software displays the results of the relative quantification calculation in the
Results table of the plate document. Figure 6-13 shows an example of the results
table containing the data from a TaqMan Human Cytokine Plate I.
s
Figure 6-13
Analysis Table of the Absolute Quantification Plate Document
The columns of the table are:
• Position – Displays the plate the sample is from and the position on the plate
(well number)
• Sample – Displays the name assigned to the well/sample when the associated
plate was run
• Detector – Displays the name of the detector assigned to the sample/well
position
• Task – Displays the task applied to the detector assigned to the sample/well
position
• CT – Displays the average calculated threshold cycle for the replicate group
associated with the test sample.
Note: The software displays Undetermined in the CT column when the
associated sample fails to produce a noticeable rise in fluorescent signal during
the entire PCR (the fluorescent signal produced by the well never crosses the
threshold level defined for the associated detector).
• ∆CT – Displays the normalized threshold cycle for the sample
• Avg∆CT – Displays the averaged normalized threshold cycle for the sample
replicate group
• ∆CT SD – Displays the number of standard deviations from the average ∆CT of
the replicate group that the sample’s individual ∆CT value lies
• ∆∆CT – Displays the calculated ∆∆CT value for the replicate group associated
with the test sample.
• RQ – Displays the calculated relative level of gene expression for the replicate
group associated with the test sample.
• RQ min – Displays the minimum calculated relative level of gene expression in
the test samples
• RQ max – Displays the maximum calculated relative level of gene expression
in the test samples
• Status – When autocalling is enabled and a sample is reject by the algorithm,
the status field indicates how the sample failed the analysis.
• Rejected – When autocalling is enabled, indicates if the sample data was rejected.
6-34
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After the Analysis
After the Analysis
User Access
Requirement
Post-Analysis
Options
Saving the
Analyzed Study
as a Multiple
Plate Document
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save analyzed data from a relative quantification study.
The following options are available after the analysis:
Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
After an analysis, the SDS software allows you to save the results as an SDS 7900HT
Multiple Plate Document (*.sdm).
To save the analysis to an SDS 7900HT Multiple Plate Document (*.sdm):
1. In the SDS software, select File > Save As.
2. In the Look in field of the Save As dialog box, navigate to and select a directory
for the software to receive the new file.
3. In the File name field, enter a file name for the file
4. Click
. The software saves the plate document to the specified directory.
Saving the Study to the SDS Enterprise Database
The SDS Enterprise Database does not store relative quantification studies
(SDS 7900HT Multiple Plate Documents).
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6-35
Chapter 6 Analyzing Real-Time Data
6-36
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Section 6.3 Dissociation Curve Analysis
Section 6.3 Dissociation Curve Analysis
In This Section
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39
Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40
Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41
Determining Tm Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44
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Chapter 6 Analyzing Real-Time Data
Overview
About
Dissociation
Curve Analysis
The Applied Biosystems 7900HT Fast Real-Time PCR System supports dissociation
curve analysis of nucleic acids using SYBR® Green 1 double-stranded DNA binding
dye chemistry. The objective of dissociation curve analysis is to accurately determine
the melting temperature (Tm) of a single target nucleic acid sequence within an
unknown PCR sample. Typical uses of dissociation curves include detection of
non-specific products and primer concentration optimization.
Employing the
SYBR Green 1
Dye
Dissociation curve analysis on the 7900HT instrument is made possible through the
use of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry
(see page D-3). Dissociation curves are commonly performed following the PCR
stage of a SYBR Green dye experiment to screen for non-specific products. To
generate the data needed to create a curve, the 7900HT instrument performs a
programmed temperature ‘ramp’ in which it slowly elevates the temperature of the
plate over several minutes. The specific binding characteristic of the SYBR Green 1
Dye permits the 7900HT instrument to monitor the hybridization activity of the
nucleic acids present in the sample. During the run, the instrument records the
decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA.
Mathematical
Transformations
After the run, the SDS software processes the raw fluorescence data from the SYBR
Green 1 Dye to generate a more meaningful representation of the relationship
between spectral change and temperature for the dissociation curve run.
Multicomponenting and Normalization
The first mathematical transformation involves the conversion of the raw data,
expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components
using the extracted pure dye standards. At the same time, the software determines the
contribution of each dye in the raw data using the multicomponent algorithm.
Afterwards, the software normalizes the data using the component of the passive
reference dye as shown below.
R ( SYBR )
R n = --------------------------------------------R ( PassiveReference )
Derivation of Dissociation Curve Data
The SDS software then computes the first derivative of the normalized data (Rn) for
each reading taken by the 7900HT instrument during the temperature ramp. The
resulting derivative data (Rn´) is the rate of change in fluorescence as a function of
temperature (see below).
dR
R n ′ = ---------n
dT
The software plots the negative of the resulting derivative data on graph of -Rn´
versus temperature (T) that visualizes the change in fluorescence at each temperature
interval. The Tm for the target nucleic acid can be determined from the graph by
identifying the maximum for the rate of change (displayed as a peak) for the
appropriate amplification curve.
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Before You Begin
Example Results
Figure 6-14 illustrates a typical dissociation curve from an experiment run to detect
non-specific amplification in cDNA samples. The figure displays the dual
amplification peaks typical of primer-dimer formation. The amplification from the
specific product is displayed with a Tm of 80.5 °C, while the primer-dimer product
has a characteristically lower Tm of 74.9 °C.
-6.0
Primer
Dimer
-5.0
Main Product
Tm = 80.5 °C
-Rn
-4.0
-3.0
-2.0
-1.0
0.0
60
65
70
75
80
85
90
95
Temperature (°C)
Figure 6-14
Example of Results from a Dissociation Curve Analysis
Before You Begin
Using the SDS
Software
Online Help
For specific instructions on any procedure described within this section, refer to the
Sequence Detection Systems Software Online Help. To get help at any time during the
procedure, click
located within the dialog box or window in which you are
working.
Examples in This
Chapter
The illustrations and screenshots that appear within this chapter were created from a
plate run to determine the purity of a β-actin amplification in unknown samples.
Each well of the plate contains SYBR Green 1 dye, forward and reverse primers, and
genomic DNA known to contain complimentary binding sites.
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Chapter 6 Analyzing Real-Time Data
Analysis Checklist
Workflow
Overview
1. Analyze the run data (see page 6-41).
2. View the results of the dissociation curve analysis (see page 6-42).
3. Determine melting temperature (Tm) values for the derivative peaks
(see page 6-44).
4. Choose from the following post-analysis options (see page 6-44):
– Reanalyze the run data.
– Adjust the display settings for the results table, plate grid, and plate
document plots.
– Print elements of the plate document.
– Export the plate document results table or plots.
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Opening the Run Data
Opening the Run Data
User Access
Requirement
Open the
Un-analyzed
Plate Document
There is no access requirement. All users can open dissociation curve data that has
been saved to the SDS Enterprise Database.
Opening a Plate Document File
1. Click
(or select File > Open).
2. In the Look in field, navigate to and select the plate document file.
3. Click
.
The SDS software displays the plate document file.
4. Configure the Analysis Settings for each marker as explained below.
Opening Un-analyzed Data from the SDS Enterprise Database
5. Click
(or select File > Open Document from Database).
6. In the Plate Query dialog box, configure the Find Plates Matching These
Criteria table with parameters that correspond to the session of interest.
7. Click
.
8. In the Search Results list, scroll to and select the session that you want to
analyze.
9. Click
10. Click
.
when finished.
Analyzing the Run Data
User Access
Requirement
Analyzing the
Run
There is no access requirement. All users can analyze dissociation curve data that has
been saved to the SDS Enterprise Database.
The run data from a temperature ramp can be analyzed immediately following the
completion of the run. For an explanation of how the software manipulates the raw
data, see “Algorithmic Manipulation of Raw Data” on page 6-7.
1. Select all wells in the plate grid.
The software outlines the selected wells with a black border.
2. Choose one of the following:
• Select Analysis > Analyze.
• Click
.
The software analyzes the run data and displays the results in the Dissociation
Curve tab.
3. Determine the Tm for the dissociation curves displayed within the Dissociation
Curve tab as explained on page 6-44.
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Chapter 6 Analyzing Real-Time Data
Determining Tm Values for the Analyzed Run
Viewing Analyzed
Dissociation
Curve Data
The SDS software displays the results of the dissociation curve analysis within the
Dissociation Curve tab of the plate document. The tab displays the analyzed data in a
graph of the negative of the derivative (-Rn´) versus temperature (T) that visualizes
the change in fluorescence at each temperature interval during the ramp.
Note: The plot displays data from the selected wells of the plate grid. If you do not
see dissociation curve data, select the wells of the plate grid containing the SYBR
Green dye reactions.
Figure 6-15 illustrates the components of the Dissociation Plot.
Dissociation Curve tab
Dissociation Plot
Dissociation curves
Tm display and slider
Step drop-down list
Plot drop-down list
Detector drop-down
Figure 6-15
Elements of the Dissociation Plot
• Dissociation plot – The plot displays data from the selected wells in the plate grid.
Note: The properties of the Dissociation Plot are adjustable. For more
information on adjusting the appearance of the plot, click the help button (
and see the Sequence Detection Systems Software Online Help.
)
• Step drop-down list – Chooses the data displayed within the plot based on the
ramp.
If a plate document contains data from more than one temperature ramp, the
Step drop-down list allows you to displays the data from each by selecting the
position of the ramp in the thermal profile.
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Determining Tm Values for the Analyzed Run
• Tm display and slider – The SDS software displays the Tm below the green slider
(see below).
Tm
There are two definitions for the Tm value:
– The chemical definition is the temperature at which 50% of the DNA is in a
double-stranded configuration.
– The mathematical definition is the maximum value for the first derivative
curve within a specific peak.
• Plot drop-down list – Chooses the data displayed within the plot based on the
derivative calculation. The list offers the following selections:
– Raw – When selected, this option plots the normalized reporter fluorescence
data (Rn) on a graph of fluorescence vs. temperature (left-most plot in
Figure 6-16).
– Derivative – When selected, this option plots derivative data (Rn´) on a graph
of the derivative vs. temperature (right-most plot in Figure 6-16). The
derivative data is the negative of the rate of change in fluorescence as a
function of temperature.
Figure 6-16 shows the plots accessible from the Plot drop-down list.
Raw Plot
Figure 6-16
Derivative Plot
Plots of the Dissociation Curve Tab
• Detector drop-down list – Chooses the data displayed within the plot based on
detector name.
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Chapter 6 Analyzing Real-Time Data
Determining
Tm Values
1. Move the pointer over the green Tm line located on the Y-axis line of the plot.
2. Click and drag the Tm line to the maximum point of the derivative plot of interest.
The SDS software displays the Tm for the curve below the Tm line.
Tm display and slider
Note: The apex of the curvature of represents the maximum rate of change in
normalized fluorescence.
After the Analysis
User Access
Requirement
Post-Analysis
Options
6-44
If using the SDS Enterprise Database, you must belong to the Scientist or
Administrator User Group to save analyzed data from a dissociation curve experiment.
Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Section 6.4 Procedure Reference
Section 6.4 Procedure Reference
In This Section
Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46
Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49
After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52
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Chapter 6 Analyzing Real-Time Data
Setting the Baseline and Threshold Values
Setting the
Baseline and
Threshold Values
for the Run
If you choose to set the baseline and threshold values manually for any detector in the
study, you need to perform the following procedure for each detector.
1. Select the Results tab.
2. If setting the baseline and threshold for a:
Absolute Quantification Plate Document
Select all wells of the plate by clicking the upper-left corner of the plate
grid.
Button
Relative Quantification Multiple Plate Document
a. Select a plate in the Plates field to view, then select all wells of the plate by
clicking the upper-left corner of the plate grid.
b. Repeat step a to select all wells of every plate in the study.
Click here to select all
wells of the plate grid
Select a plate here
3. In the Detector drop-down list in the Amplification plot, select a detector for
which you want to set the baseline and threshold values.
The software displays the data for the selected wells within the Amplification plot.
4. In the Plot drop-down list, select ∆Rn vs. Cycle.
5. Double-click the Amplification plot, or click
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Setting the Baseline and Threshold Values
6. In the Display Settings dialog box:
a. In the Select Pane/View field, select Amplification Plot.
b. In the Y Axis group box of the Scale tab, select the Linear option button.
c. Click
.
7. Identify the components of the linear scale Amplification plot and set the
baseline so that the amplification curve growth begins at a cycle number greater
than the Stop baseline cycle.
If you are inspecting an AutoCalled detector, verify that the baseline is called
correctly by the software and proceed to step 8. If the baseline is set incorrectly,
open the Analysis Settings dialog box, configure the detector for manual
calling, then click
. After the software adjusts the data, manually set the
baseline and threshold for the detector as described in this procedure.
If the amplification plot looks like...
Then...
The amplification curve begins after the
default maximum baseline (cycle 15).
Adjustment of the baseline is not
required.
The maximum baseline is set too high.
Decrease the Stop baseline value by
dragging the right range marker ( ) to
an earlier cycle.
The maximum baseline is set too low.
Increase the Stop baseline value by
dragging the right range marker ( ) to a
later cycle.
8. Double-click the Amplification plot or click
.
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Chapter 6 Analyzing Real-Time Data
9. In the Display Settings dialog box:
a. In the Select Pane/View field, select Amplification Plot.
b. In the Y Axis group box of the Scale tab, select the Log option button.
c. Click
.
10. Identify the components of the amplification curve and set the threshold so that
it is:
• Above the background
• Below the plateaued and linear regions of the amplification curve
• Within the geometric phase of the amplification curve
If you are inspecting an AutoCalled detector, verify that the threshold is called
correctly by the software and proceed to step 11. If the threshold is set
incorrectly, open the Analysis Settings dialog box, configure the detector for
manual calling, then click
. After the software adjusts the data, manually
set the baseline and threshold for the detector as described in this procedure.
Plateau phase
Linear phase
Geometric phase
Threshold setting
(click and drag)
Rn
Background
Cycle
Baseline
11. Repeat steps 3 through 10 to set the baseline and threshold values for all
remaining detectors present on the plate.
12. If you choose to eliminated outliers
• Automatically – View the results.
• Manually – Visualize and eliminate outliers from the analyzed run data as
explained in “Eliminating Outlying Amplification” on page 6-49.
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Eliminating Outlying Amplification
Eliminating Outlying Amplification
Overview
For any PCR, experimental error may cause some wells to amplify insufficiently or not
at all. These wells typically produce CT values that differ significantly from the average
for the associated replicate wells. If included in the absolute or relative quantification
calculations, these outliers can potentially result in erroneous measurements.
Visualizing
Outliers
To ensure precise relative quantification, replicate groups must be carefully
examined for outlying wells. The CT vs. Well Position view of the Amplification plot
allows you to examine each set of replicate wells for outliers.
1. Select the Results tab.
2. If analyzing a Relative Quantification study, select a plate in the Plates field and
deselect all wells in the plate grid. Repeat for each plate in the Plates field.
When you are finished, the Amplification plot should not display any data.
3. If eliminating outliers from a:
Absolute Quantification Plate Document
Select all wells of the plate by clicking the upper-left corner of the plate grid.
Relative Quantification Multiple Plate Document
a. Select a plate in the Plates field to view.
b. Select all wells of the plate by clicking the upper-left corner of the plate grid.
4. In the Plot drop-down list, select Ct vs. Well Position.
5. In the Well versus Threshold Cycle Amplification plot, verify the uniformity of
each replicate population by comparing the groupings of CT values for the wells
that make up the set.
Are outliers present?
Then…
Yes
Record the well numbers of the outlying wells, then go to
the next step.
No
Go to the next step.
6. In the Detector drop-down list, select another detector, and repeat steps 3
through 5. Repeat until each detector has been screened for outliers.
7. If analyzing a Relative Quantification Multiple Plate Document, deselect the
wells of the current plate, then repeat steps 3 through 6 for every plate in the
study.
8. Are outliers present on the plate?
Yes – If outliers are present for any detector, eliminate the outlying wells from
the analysis by omitting them as explained in “Eliminating Wells from the
Analysis” on page 6-50.
No – If no outliers are present, do one of the following:
• If analyzing an Absolute Quantification Plate Document, view the results of
the run displayed within the Standard Curve plot and Results table.
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Chapter 6 Analyzing Real-Time Data
• If analyzing a Relative Quantification Multiple Plate Document, view the
results of the run displayed within the Gene Expression plot and
Results table.
Eliminating Wells
from the Analysis
The SDS software allows you to omit wells from the analysis. If you have identified
outliers or data you want to exclude from the analysis, follow the procedure below to
remove the corresponding wells from use.
IMPORTANT! If a well is removed from use before the plate document is run, the
SDS software does not collect data for the well during the run. However, if a well is
removed from use after the plate document has been run, the software excludes the
well from the analysis but does not delete the data.
1. If omitting wells from a Relative Quantification Multiple Plate Document,
select a plate in the Plates field that contains wells you want to omit.
2. While pressing and holding the Ctrl key, select the well(s) in the plate grid that
you want to omit.
3. If omitting wells from a Relative Quantification Multiple Plate Document,
repeat steps 1 through 2 for each plate in the study.
4. Select the Setup tab.
5. In the well inspector, click the Omit Well(s) check box to exclude the selected
wells from the analysis.
Selected wells
(removed from
use)
Omit Well(s) check
box (selected)
Note: To reinstate an omitted well, select the well, then click the Omit Well(s)
check box.
6. Click
(or select Analysis > Analyze).
7. Repeat the procedures in “Visualizing Outliers” on page 6-49 to verify that all
outliers have been removed from the analysis.
6-50
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After the Analysis
After the Analysis
User Access
Requirement
Post-Analysis
Options
Changing the
Display Settings
If using the SDS Enterprise Database, all users can perform the tasks explained
below except for saving the results. Only users belonging to the Scientist or
Administrator User Group can save analyzed data to the database as a session.
Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)
Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-53
Before printing or exporting the analyzed data, you can reconfigure the appearance
of several elements of the plate document including the results table, plate grid, and
most plots.
1. Click
(or select View > Display Settings).
2. In the Display Settings dialog box, click
modifying the display settings.
Printing a Report
The SDS software can print a report of the analyzed data containing individual or
multiple elements of the plate document.
1. Click
(or select File > Print Report).
2. In the Print Report dialog box, click
previewing, and printing the report.
Exporting Plate
Document Data
for further instructions on
for instructions on setting up,
Exporting Plate Document Data as a Tab-Delimited Text File
The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for
all or a select group of wells on a plate document. The exported files are compatible
with most spreadsheet applications and programs that can read tab-delimited text.
To export run data as a tab-delimited text file, choose one of the following for further
instructions:
• See “Exporting Plate Document Data” on page A-16.
• Click
within the table view.
Exporting Plots as Graphics
The SDS software can export most panes and plots of the plate document as JPEG
(Joint Photographic Experts Group) graphic files. The JPEG file format is
compatible with most word processing and spreadsheet applications and can be
incorporated directly into HTML documents for viewing by most web browser
software.
To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or click
in the plot of interest for further instructions.
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Chapter 6 Analyzing Real-Time Data
Saving the
Results
The SDS software saves the results of the analysis differently depending on whether
the analysis is saved to the SDS Enterprise Database (see below) or to a plate
document file (see page 6-54).
Saving the Analysis Session to the SDS Enterprise Database
If using the SDS Enterprise Database, you can save your analysis of the run data to
the database as an analysis session.
IMPORTANT! Observe the following when saving sessions to the database:
• If you modified the plate document information in any way (by removing wells
from use, or by changing the display settings), you must save the plate document
to the database for the changes to persist (select File > Save Plate Document to
Database).
• The SDS Enterprise Database allows two or more users to open and modify data
simultaneously. If another user has opened, analyzed, and saved the same data
that you have just analyzed, the software overwrites their analysis when you save
yours to the database.
• Analysis session results are directly linked to the configuration of the plate
document used to create the session. If, after you have saved your analysis
session to the database, you modify the linked plate document (by omitting
wells, altering sample names, etc.) your analysis session may be 'orphaned' (i.e.,
have no relationship to its attached plate document). Consequently, the results of
your analysis session may change if you reanalyze it because you have changed
the content of the associated plate document containing the raw data.
Applied Biosystems recommends the following guidelines for using analysis
sessions:
– Save the plate document as a new document any time you make changes to the
plate setup (omit wells, alter sample names, modify detectors or markers, etc.)
– Maintain only one saved analysis session per document
– Understand that only results sessions saved after the latest changes to a plate
setup are “guaranteed” to correspond to the linked plate document.
To save the save the analysis session to the database:
1. Select File > Save Results to Database.
2. In the Description field of the Save Results to Database dialog box, enter a brief
description of the plate document (up to 255-characters).
Note: The software automatically populates the Session Name field with the
name of the associated plate document.
3. (Optional) In the Add to Study field, click
, then select an existing study,
or create a new study by clicking
, and doing the following:
a. In the Name field of the Configure the Create New Study dialog box, enter
a name for the study (up to 128 characters).
The Creator field is not editable and displays your user name.
b. In the Description field, enter a brief description of the study (up to
255 characters).
6-52
c. Click
.
d. Click
.
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After the Analysis
4. In the Save Results to Database dialog box, click
5. In the confirmation dialog box, click
.
.
Saving the SDS 7900HT Document
Although the SDS software saves the display settings and analysis settings to the
plate document, it does not save the results of the analysis. You must analyze the
plate document each time you want to review the analysis.
1. In the SDS software, select File > Save As.
2. In the Look in field of the Save As dialog box, navigate to and select a directory
for the software to receive the new file.
3. In the File name field, do one of the following:
• Enter a file name for the plate document file.
• Enter or scan the bar code number for the plate into the field.
Note: The SDS software does not require that the file name match the bar code
of the corresponding plate.
4. Click
. The software saves the plate document to the specified directory.
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Chapter 6 Analyzing Real-Time Data
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Maintaining the Instrument
In This Chapter
7
7
Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2
Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3
Section 7.1 Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . 7-5
Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6
Changing the Plate Adapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12
Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14
Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16
Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20
Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27
Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30
Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35
Automation Accessory Components and Stack Positions. . . . . . . . . . . . . . . . . . . 7-36
Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . 7-37
Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41
Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49
Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52
Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . 7-53
General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54
Maintaining the SDS software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55
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Chapter 7 Maintaining the Instrument
Notes for Database Users
Database Alerts
and Notifications
If you are using an SDS Enterprise Database to store SDS plate documents, sessions,
studies, and data, you may be required to perform additional tasks when performing
the procedures described in this chapter. This section describes the types of actions
you may need to perform depending on how your administrator has configured the
database. For more information on any of the features described below, see the SDS
Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCR
System Administrators Guide.
Reason(s) For
Change Dialog
Box
The Reason(s) for Change dialog box appears only if the SDS Enterprise Database is
configured for auditing. The auditing system monitors the creation, modification,
and deletion of the SDS data contained by the database.
When the Reasons for Change dialog box appears, do one of the following:
• Select a description for the change from the drop-down list, then click
• Enter a description of the reason for the change in the field, then click
.
.
Reasons for change
drop-down list
Default descriptions
Custom description of
the reason for the change
Figure 7-1
Electronic
Signature
Verification
Dialog Box
Reasons for Change Dialog Box Options
The Electronic Signature Verification dialog box appears only if the SDS Enterprise
Database is configured to require electronic signatures. The electronic signature
system restricts and tracks user access of the SDS data contained by the database.
When the Electronic Signature Verification dialog box appears:
1. In the User ID and Password fields, enter your user name and password, then
click
.
2. After the software validates your user name and password, it displays a message
stating the success or failure of the signature. Click
to continue.
Description of the action
that requires a signature
Enter your user name
Enter your password
Figure 7-2
7-2
Electronic Signature Verification Dialog Box Options
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Recommended Maintenance Schedule
Recommended Maintenance Schedule
Maintenance
Schedule
The following table includes a list of tasks that should be performed on the
Applied Biosystems 7900HT Fast Real-Time PCR System regularly.
Table 7-1
Maintenance Schedule for the 7900HT Instrument
Interval
Task
Su M T W Th F S
See Page
Archive SDS Files
7-54
Perform a Background Run
7-16
Check (and if necessary replace) Gripper Finger
Pads
7-52
Defragment the Computer Hard Drive
7-54
Perform a Pure Dye Run*
7-20
Check Applied Biosystems Web Site for Software
Updates
7-55
Week (7 Days)
Su M T W Th F S
Month (30 Days)
January
July
Su M T W Th F S
Su M T W Th F S
Semi-Annually
(6 Months)
*Perform a background run before each Pure Dye Run.
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Chapter 7 Maintaining the Instrument
7-4
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Section 7.1 Maintaining the 7900HT Instrument
Section 7.1 Maintaining the 7900HT Instrument
In This Section
Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6
Changing the Plate Adapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12
Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14
Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16
Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20
Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27
Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30
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Chapter 7 Maintaining the Instrument
Replacing the Sample Block
When to Perform
IMPORTANT! Before changing the sample block, perform all required upgrades to
the SDS software and instrument firmware. Failure to update the software can make
the instrument inoperable or result in damage to instrument components.
You need to remove the 7900HT instrument sample block when you:
• Decontaminate the wells of the sample block (see page 7-14)
• Change sample block formats
IMPORTANT! Always run a background plate after installing the sample block.
Sample Block
Installation
Workflow
Unless instructed to do otherwise, adhere to the following guidelines when
exchanging sample block modules of different formats.
To install a sample block module:
1. Perform all required software and firmware upgrades to the Applied Biosystems
7900HT Fast Real-Time PCR System (see page 7-55).
2. Remove the existing sample block (see page 7-7).
3. Install the new sample block (see page 7-10).
4. Change the plate adapter (see page 7-12).
5. Run a background plate to check the sample block for contamination
(see page 7-16).
If you are changing block formats or installing a new block, also do the following:
IMPORTANT! Perform pure dye and background runs after replacing the sample
block, even if you are replacing a sample block module with another block of the
same format.
6. Run a pure dye plate or microfluidic pure dye card to create the pure spectra
calibration values for the new format (see page 7-20).
7. Run the appropriate TaqMan® RNase P Instrument Verification Plate or a
TaqMan® Low Density 7900HT Installation Array (TGF-β card) to confirm the
proper operation of the sample block (see page 7-30).
If you are using an Automation Accessory, also do the following:
8. If changing consumable formats (for example, when replacing a Standard
384-Well Block with a Standard 96-Well Block, a Fast 96-Well Block, or a
7900HT System TaqMan® Low Density Array Upgrade), adjust the plate sensor
switch on the Plate Handler arm for the new plates (see page 7-37).
9. Align the Zymark® Twister Microplate Handler fixed-position bar code reader
for the new plate format (see page 7-41).
IMPORTANT! You must align the Plate Handler to only the Instrument position
(Zymark position 2) as explained on pages 7-41 to 7-44.
10. Align the fixed-position bar code reader for the new plate format
(see page 7-49).
7-6
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Replacing the Sample Block
Materials
Required
Handling the
Sample Block
• Replacement Sample Block (if replacing the sample block)
• 5/32 inch hex key (necessary only for certain instruments)
• 5/16 inch hex key (some instruments may require a crescent wrench)
The interchangeable sample blocks are delicate pieces of equipment containing
several fragile components that can break if handled improperly. Figure 7-3 shows
the correct locations for handling the interchangeable sample block module.
Circuitry and connections to
the instrument (Do Not Touch)
GR2028
Heat sinks
(Bottom of module)
Figure 7-3
Removing the
Sample Block
Hold sample block
module from the sides
Locations for Handling Sample Block Modules
1. Start the Automation Controller Software, then:
a. Select the Thermal Status tab, then confirm the function of the current
module. The module is operating normally if the software is receiving a
temperature reading.
b. Click
to rotate the instrument tray to the OUT position.
2. Select File > Exit to close the Automation Controller Software.
3. Power off and unplug the 7900HT instrument.
PHYSICAL HAZARD. The instrument must be unplugged
and powered off at all times during the following procedure. Failure to comply
can result in serious physical injury to the user or damage to the instrument.
4. Wait 20 to 30 min for the heated cover to cool.
PHYSICAL HAZARD. During instrument operation, the
temperature of the sample block can be as high as 100 °C. Before performing
this procedure, wait until the sample block reaches room temperature.
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7-7
Chapter 7 Maintaining the Instrument
5. If the instrument tray is in the OUT position (outside of the instrument), push it
into the instrument to provide an open workspace.
6. If using a Zymark Twister Microplate Handler, remove the covers for the
fixed-position bar code reader and the underlying platform.
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-position bar code reader
and underlying platform covers
7. Push the instrument tray inside the instrument, then remove the thermal cycler
access cover to permit access to the sample block.
GR2023b
IMPORTANT! The thermal cycler access cover is secured to the instrument by
non-locking pins and may require force to remove it (no tools are required).
Access cover
8. Using a 5/16-inch hex key, turn the sample block locking bolt counterclockwise until it is very loose but still attached to the sample block locking bar.
GR2024
IMPORTANT! Some instruments may require the use of an adjustable crescent
wrench to loosen the sample block locking bolt.
Sample block
locking bar
Sample block
locking bolt
7-8
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Replacing the Sample Block
GR2024b
GR2025b
9. Loosen the thumbscrew securing the sample block locking bar to the instrument
chassis (may be a 5/32-inch hex bolt on some instruments).
thumbscrew
GR2025b
10. Lift the sample block locking bar up and out of the instrument.
11. Remove the sample block from the instrument:
a. Rotate the release lever at the base of the sample block 90 degrees.
GR2027
b. Being careful not to damage the heat sinks on the bottom of the sample
block, slide the sample block out of the instrument and place it on a clean,
level surface.
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7-9
Chapter 7 Maintaining the Instrument
Replacing the
Sample Block
IMPORTANT! Before changing the sample block, perform all required upgrades to
the SDS software and instrument firmware. Failure to upgrade the software can
render the instrument inoperable or result in damage to instrument components.
1. Load the sample block into the instrument compartment:
a. Being careful not to damage the heat sinks on the bottom of the sample
block, rest the sample block on the metal runners on either side of the
instrument bay.
b. Carefully slide the sample block into the instrument until the front of the
block is flush with the rear of the locking bar.
GR2027
c. After it is seated, firmly press on the sample block to ensure a good
connection.
GR2025b
2. Replace the sample block locking bar.
3. Tighten the thumbscrew (from step 9 on page 7-9) to secure the sample block
locking bar to the instrument chassis (may be a 5/32-inch hex bolt).
4. Using the 5/16-inch hex key, turn the sample block locking bolt clockwise until
it is flush with the locking bar.
5. Again, press on the right and left sides of the front surface of the sample block
to ensure that it is seated securely.
6. Replace the thermal cycler access cover:
a. Fit the lip at the bottom of the access cover over the lower edge of the bay.
b. Push the cover towards the instrument until it snaps into place.
IMPORTANT! You must reinstall the thermal cycler access cover before powering
GR2023b
on the instrument. Failure to do so prevents the instrument from uploading the
firmware from the computer and causes the SDS software to display an error.
7-10
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Replacing the Sample Block
7. If using a Plate Handler, replace the covers for the fixed-position bar code
reader and the underlying platform (removed in step 6 on page 7-8).
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-position bar code reader
and underlying platform covers
8. Plug in and power on the 7900HT instrument.
9. Confirm the function of the installed sample block module:
a. Start the Automation Controller Software.
b. Select the Thermal Status tab.
Does the software
display temperatures?
Yes
Then…
the installation is successful.
The presence of temperature readings confirm that the
7900HT instrument successfully established the
connection to the new sample block.
No
the 7900HT instrument is unable to establish
communication with the new sample block.
To troubleshoot the problem:
1. Power off and unplug the 7900HT instrument.
2. Remove the thermal cycler access cover.
3. Press on the right and left sides of the front plate of
the sample block to ensure that it is seated securely.
4. Reinstall the thermal cycler access cover.
5. Repeat step 8 until you hear a high-pitched tone
confirming communication between the instrument
and sample block.
10. After the sample block is loaded into the instrument do the following:
a. Perform a background run (see page 7-16) to verify that the sample block:
– Is connected and working properly
– Contains no contaminants that will interfere with fluorescent detection
b. If changing sample block formats, perform any remaining tasks outlined in
the “Sample Block Installation Workflow” on page 7-6.
IMPORTANT! Perform pure dye and background runs after replacing the sample
block, even if you are replacing a sample block module with another block of the
same format.
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7-11
Chapter 7 Maintaining the Instrument
Changing the Plate Adapter
When to Perform
Remove and replace the 7900HT instrument plate adapter after changing the sample
block module format (for example, replacing a Standard 384-Well Block with a
Fast 96-Well Block).
Note: The sample block must be used with the corresponding plate adapter of the
same plate format.
Materials
Required
Changing the
Plate Adapter
• 3/32-inch hex key
• One of the following:
– 384-Well Plate Adapter
– 96-Well Plate Adapter
– Fast 96-Well Plate Adapter
– TaqMan® Low Density Array Adapter
1. If the instrument tray is inside the 7900HT instrument, move the instrument tray
to the OUT position:
a. Start the SDS software.
b. Click
(or select File > New).
c. In the New Document dialog box, click
.
d. In the new plate document, select the Instrument tab.
e. In the Real-Time tab of the Instrument tab, click
The instrument tray rotates to the OUT position.
.
f. Select File > Exit. The SDS software exits.
2. Remove the four screws attaching the plate holder to the plate arm.
Unscrew
Unscrew
3. Remove the plate adapter from the instrument tray.
Note: If changing sample block formats (for example, replacing a Standard
384-Well Block with a Fast 96-Well Block), store the plate adapter with the
sample block module of the same format.
7-12
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Changing the Plate Adapter
4. Place the new plate adapter into the instrument tray with the A1 label in the
rear-left corner (see below).
IMPORTANT! Make sure to install the correct version of the plate adapter for
the plate format you intend to use. The plate adapters are labeled for the
consumable format they support.
Well A1
Label
5. Replace and tighten the four screws in the order shown below:
IMPORTANT! The order in which the screws are tightened is important to
ensure proper alignment of the plate to the sample block inside the 7900HT
instrument.
3
1
2
4
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7-13
Chapter 7 Maintaining the Instrument
Decontaminating the Sample Block
When to Perform
The following procedure describes how to decontaminate the wells of a sample block
module. The procedure eliminates residual PCR-related products, including
fluorescent labeled TaqMan® probes. Clean the sample block as often as needed.
IMPORTANT! If you plan to use a cleaning or decontamination method other than
the one in this manual, check with Applied Biosystems first to ensure that the
method will not damage the sample block module or the 7900HT instrument.
Materials
Required
•
•
•
•
10% Sodium hypochlorite (bleach) solution
Isopropanol, 100 percent pure
5/32 in hex key
Cotton swabs
CHEMICAL HAZARD. Sodium hypochlorite (bleach) is a
liquid disinfectant that can be corrosive to the skin and can cause skin
depigmentation. Please read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
CHEMICAL HAZARD. Isopropanol is a flammable liquid and
vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or
repeated contact may dry skin and cause irritation. It may cause central nervous
system effects such as drowsiness, dizziness, and headache, etc. Please read the
MSDS, and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
Cleaning a Low
Density Array
Block
This procedure applies to the sample block on the 7900HT System TaqMan Low
Density Array Upgrade.
1. Wipe the surface of the sample block (grey aluminum) with 10% bleach.
2. Wipe the sample block three times with distilled water.
3. Wipe the sample block with isopropanol, then allow to air dry.
Circuitry and connections to
the instrument (Do Not Touch)
Warning
GR2028
Well A-1
4. Replace the block as explained in “Replacing the Sample Block” on page 7-10.
5. Run a background Low Density Array to confirm that the contamination has
been removed.
7-14
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Decontaminating the Sample Block
Cleaning a
Sample Block
This procedure applies to a:
• Standard 384-Well Block
• Standard 96-Well Block
• Fast 96-Well Block
1. Remove the sample block from the 7900HT instrument as explained in
“Removing the Sample Block” on page 7-7.
2. Use the following figure as a guide to locate the suspected contaminated wells
on the sample block.
Circuitry and connections to
the instrument (Do Not Touch)
Warning
GR2028
Well A-1
3. Pipette the appropriate volume of 10% bleach solution into each suspected
contaminated well of the sample block module.
• For a Standard 384-Well Block, pipette 40 µL bleach solution to each well.
• For a Standard 96-Well Block, pipette 150 µL bleach solution to each well.
• For a Fast 96-Well Block, pipette 55 µL bleach solution to each well.
4. Allow the sample block to sit for 3 to 5 min.
5. Using a pipet, remove the bleach solution from the wells of the sample block.
6. Rinse (pipette and remove) each contaminated well with three treatments of
deionized water at the appropriate volume for the sample block.
• For a Standard 384-Well Block, rinse affected wells with 40 µL deionized
water.
• For a Standard 96-Well Block, rinse affected wells with 150 µL deionized
water.
• For a Fast 96-Well Block, rinse affected wells with 55 µL deionized water.
Note: Absolute isopropanol can be substituted for water in the third treatment.
7. Remove any remaining isopropanol or water from the wells of the sample block
module.
8. Replace the sample block as explained in “Replacing the Sample Block” on
page 7-10.
9. Run a background plate to confirm that the contamination has been removed.
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7-15
Chapter 7 Maintaining the Instrument
Performing a Background Run
When to Perform
Applied Biosystems recommends running a background plate monthly or as often as
necessary depending on instrument use.
Purpose of
Background Runs
A background run measures the ambient fluorescence in a background plate or Low
Density Array containing deionized water. During the run, the 7900HT instrument
performs a continuous scan of the plate for 2 minutes at 60 °C. Afterwards, the SDS
software averages the spectrum recorded during the run and extracts the resulting
spectral component to a calibration file. The software uses the calibration file during
subsequent runs to remove the background signal from the run data.
Because the background signal can change with instrument age, Applied Biosystems
recommends regenerating the Background component calibration every month.
Note: Background runs can also be used to detect and troubleshoot sample block
contamination.
About the
Background
Component
Materials
Required
7-16
Fluorescence collected by the Applied Biosystems 7900HT Fast Real-Time PCR
System includes a background component, a fluorescent signal that is inherent to the
system. The background component is a composite signal found in all spectral data
that consists of fluorescence from several sources including: the background
electronic signal, the sample block, the water inside the consumable, and the plastic
consumable itself. Because the background signal can interfere with the precision of
SDS data, the 7900HT instrument has been engineered to minimize the background
signal. Additionally, the SDS software also algorithmically eliminates the
background signal from each fluorescent sample to maximize the instrument’s
sensitivity.
One of the following:
• Background plate included in a Spectral Calibration Kit and a centrifuge with
plate adapter
• Components for making a standard 384- or 96-well background plate:
– ABI PRISM™ 394- or 96-Well Optical Reaction Plate
– An optical adhesive cover or ABI PRISM™ Optical Flat Caps
– Pipettor, 100-µL (with pipet tips)
– Centrifuge, with plate adapter
• Components for making a TaqMan Low Density Array background plate:
– Microfluidic card spectral calibration reagents
– Centrifuge
– Microfluidic card sealer
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Performing a Background Run
Preparing the Background Plate or TaqMan Low Density Array
Preparing a
Background Card
1. Remove the background Low Density Array from its packaging and set it aside
to warm to room temperature.
2. Remove the tube of background solution from the freezer to thaw. When the
solution has thawed, vortex the tube.
3. Load the background solution into the background Low Density Array, loading
100 µL of solution per fill reservoir.
4. Centrifuge the Low Density Array according to the procedures in “Centrifuging
the TaqMan Low Density Arrays” on page 4-16.
5. Seal the Low Density Array according to the procedures in “Sealing the
TaqMan Low Density Arrays” on page 4-19.
6. Continue with “Creating a Plate Document for the Background Run” below.
Preparing and
Running a
Background Plate
1. Choose from the following:
If…
Then…
using a background
plate from a Spectral
Calibration Kit
remove the plate from the freezer and allow it to thaw to
room temperature.
creating a
background plate
1. Pipette deionized water to each well of the plate.
– If using a standard 384-well plate, add 20 µL per well.
– If using a standard 96-well plate, add 50 µL per well.
2. Seal the plate using an optical adhesive cover or optical
flat caps.
2. Briefly centrifuge the background plate.
3. Continue with “Creating a Plate Document for the Background Run” below.
Creating a Plate Document for the Background Run
Preparing a
Background Plate
Document
1. Start the SDS software.
2. Click
(or select File > New).
3. Configure the New Document dialog box:
• Assay – Select Background.
• Container – Select the appropriate plate format.
• Template – Select Blank Template.
4. If the background plate or Low Density Array is labeled with a bar code, click
the Barcode field, then scan the bar code number using the hand-held bar code
reader.
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7-17
Chapter 7 Maintaining the Instrument
5. Click
.
The software creates a plate document with the attributes for a background run.
IMPORTANT! Do not modify the background plate document. The method for a
Background run is coded into the SDS software and consists of a single hold at
60 °C for 2 min. Because the plate contains only deionized water, the plate
document does not require sample or detector labels.
6. Save the plate document:
a. Click
(or select File > Save).
b. In the File name field of the Save dialog box, enter:
Background_<date in MMDDYY format>
For example, the file name for a background plate run on May 31, 2001
would be: Background_053101.
c. Click
.
The software saves the plate document and is now configured for the
Background run.
7. Continue with “Running the Prepared Background Plate or TaqMan Low
Density Array” below.
Running the Prepared Background Plate or TaqMan Low Density Array
Performing the
Background Run
1. Load the background plate into the 7900HT instrument:
a. In the plate document in the SDS software, select the Instrument tab.
b. In the lower portion tab, select the Real-Time tab.
c. In the Real-Time tab of the Instrument tab, click
.
d. Place the background plate or Low Density Array into the instrument tray
as shown below.
Well A1
Position the plate so that the bar code
faces towards the front of the instrument
Well A1
Position the Low Density Array so that the bar
code faces towards the front of the instrument
Note: The A1 position is in the top-left side of the instrument tray.
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Performing a Background Run
2. Click
.
The 7900HT instrument begins the background run.
Note: Before starting the run, the instrument may pause (up to 15 min) to heat
the heated cover to the appropriate temperature.
3. When the background run is complete and the Run Complete dialog box
appears:
a. Click
to close the dialog box.
b. Click
and remove the plate from the instrument tray.
c. Extract the background component as explained on page 7-19.
Analyzing the Background Data
Extracting the
Background
In this procedure, you will extract the calibration values from the background plate
document. After extraction, the SDS software stores the data as part of the calibration
file located in the Calibration subdirectory of the SDS directory.
1. Select Analysis > Extract Background.
The software attempts to extract the background signal and displays the success
of the extraction in a dialog box.
If the software displays the:
• Calibration Update Complete dialog box, then the run is successful.
Proceed to step 2.
The raw spectra read from the Background plate conform to acceptable
limits.
• Error dialog box, then the run is unsuccessful. Troubleshoot and
decontaminate the sample block as explained in “Background Runs” on
page 8-9.
The software has stopped the extraction because one or more raw spectra
exceed 2500 FSU.
2. Click
(or select File > Save).
The software saves the plate document.
3. Select File > Close.
The software closes the plate document.
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7-19
Chapter 7 Maintaining the Instrument
Performing a Pure Dye Run
When to Perform
Applied Biosystems recommends performing spectral calibration:
• Every 6 months depending on instrument use
• After changing sample block formats (see page 7-6)
IMPORTANT! Always run a background plate before performing a Pure Dye
calibration.
Purpose of Pure
Dye Runs
Pure dye data is generated from the results of a pure dye run in which the SDS
software collects spectral data from a set of dye standards during a 2-min hold at
60 °C. The software stores the spectral information for the pure dye standards in a
calibration file located in the SDS directory. After the run, the software extracts each
component dye spectrum from the collected data in the pure spectra run file.
IMPORTANT! Because the age and use of instrument components can affect pure
spectra readings, Applied Biosystems recommends updating the pure spectra data
files once or twice annually depending on instrument use.
Components of
the Pure Dye
Spectra
The Applied Biosystems 7900HT Fast Real-Time PCR System monitors fluorescent
signals generated by several dyes inside the preloaded pure dye plate. For the
standard 384-well, standard 96-well, or Fast 96-well pure dye plates, the dyes are:
FAM™, NED™, ROX™, SYBR® Green, TAMRA™, TET™, and VIC®.
For the 7900HT System TaqMan Low Density Array Upgrade, the 7900HT
instrument monitors fluorescent signals generated by three dyes: FAM™, VIC®, and
ROX™. Your installation kit contains these dyes, and you introduce them into three
separate microfluidic pure dye cards to perform pure dye runs.
1 2 3
Figure 7-4
4
5 6
7
Dye
Peak (nm)
1
FAM
~520
2
SYBR Green
~520
3
TET
~540
4
VIC
~550
JOE
~550
5
NED
~570
6
TAMRA
~580
7
ROX
~610
Standard Pure Dye Spectra of the 7900HT Instrument
Note: The 7900HT instrument supports the detection of custom pure dyes (dyes
other than those provided by Applied Biosystems). To add custom pure dyes to the
Pure Dye set for your instrument, see “Adding Custom Dyes to the Pure Dye Set” on
page 7-27.
7-20
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Performing a Pure Dye Run
After a run, the SDS software receives run data in the form of a raw spectra signal for
each reading. To make sense of the raw data, the software must determine the
contribution of each fluorescent dye used in the sample through a process called
multicomponenting. The software accomplishes the separation by comparing the raw
spectra with a set of pure dye standards contained in a calibration file. When a plate
document is saved after analysis, the software stores the pure spectra information
with the collected fluorescent data for that experiment.
Materials
Required
384- or 96-Well Plate
• Appropriate Spectral Calibration Kit:
– Sequence Detection Systems 384-Well Spectral Calibration Kit
– ABI PRISM™ 7900HT Sequence Detection Systems 96-Well Spectral
Calibration Kit
– 7900HT System Fast 96-Well Spectral Calibration Kit
Note: Both the standard 96-well and Fast 96-Well Spectral Calibration Kits
contain two pure dye plates.
• Product Insert
• Centrifuge, with plate adapter
Microfluidic Pure Dye Cards
• Microfluidic card spectral calibration reagents
• Centrifuge
• Microfluidic card sealer
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7-21
Chapter 7 Maintaining the Instrument
Preparing the Pure Dye Plates or Cards
Preparing the
Pure Dye Plates
IMPORTANT! A background run must be performed before running a pure dye plate.
See “Performing a Background Run” on page 7-16 for more information.
1. Remove the pure dye plate from the refrigerator or freezer and set it aside to
warm to room temperature.
2. Remove the pure dye plate from its packaging.
3. Briefly centrifuge the pure dye plate.
Preparing the
Microfluidic Pure
Dye Cards
IMPORTANT! A background run must be performed before running the microfluidic
pure dye cards. See “Performing a Background Run” on page 7-16 for more
information.
1. Remove the three empty microfluidic pure dye cards from the refrigerator and
set them aside to warm to room temperature.
2. Remove the three dye tubes (FAM, VIC, and ROX dyes) from the freezer to
thaw. When the dyes have thawed, vortex the tubes.
3. Load the FAM dye into one of the empty microfluidic pure dye cards, loading
100 µL of dye per fill reservoir.
4. Centrifuge the microfluidic pure dye card per the procedures in “Centrifuging
the TaqMan Low Density Arrays” on page 4-16.
5. Seal the microfluidic pure dye card per the procedures in “Sealing the TaqMan
Low Density Arrays” on page 4-19.
6. Repeat steps 3 through 5 for the VIC and ROX dyes.
Preparing a Plate Document for a Pure Dye Plate or Card
Preparing a
Pure Dye Plate
Document
1. Start the SDS software.
2. Click
(or select File > New).
3. Configure the New Document dialog box:
Assay – Select Pure Spectra.
Container – Select the appropriate format.
Template – If performing a:
• Standard 384-Well Pure Dye Run: select 384 Well Pure Dyes Plate.sdt.
If no plate document templates are available, construct a Pure Dye plate
document using the product insert from the Spectral Calibration Kit and
the procedure on page 7-28.
• Standard 96-Well Pure Dye Run: select the plate document template
matching the Pure Dye plate you intend to run.
To run Plate 1 (containing FAM, JOE™, NED, and ROX dyes), select
96 Well Pure Dyes Plate 1.sdt.
To run Plate 2 (containing SYBR, TAMRA, TET, and VIC dyes), select
96 Well Pure Dyes Plate 2.sdt.
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Performing a Pure Dye Run
• Fast 96-Well Pure Dye Run: select the plate document template matching
the Pure Dye plate you intend to run.
To run Plate 1 (containing FAM, JOE, NED, and ROX dyes), select
Fast 96 Well Pure Dyes Plate 1.sdt.
To run Plate 2 (containing SYBR, TAMRA, TET, and VIC dyes), select
Fast 96 Well Pure Dyes Plate 2.sdt.
• Microfluidic Pure Dye Run: leave the field blank.
To run the microfluidic pure dye card containing FAM dye,
select MFC FAM Pure Dyes.sdt.
To run the microfluidic pure dye card containing ROX dye,
select MFC ROX Pure Dyes.sdt.
To run the microfluidic pure dye card containing VIC dye,
select MFC VIC Pure Dyes.sdt.
4. In the Barcode field, scan the bar code number using the hand-held bar code
reader.
5. Click
. The software displays a plate document with the attributes for a
pure dye run.
6. Save the Pure Dye plate document:
a. Click
(or select File > Save).
b. Select Files of type > SDS 7900HT Document (*.sds).
c. In the File name field, if running a:
• Standard 384-Well Plate, enter PureDye_<date in MMDDYY format>
For example, the file name for a plate run on May 31, 2001 would be:
PureDye_053101.
• Standard 96-Well Plate, enter PureDye_Plate<plate #>_<date in
MMDDYY format>
For example, the file name for a Pure Dye Plate 1 run on May 31, 2001
would be: PureDye_Plate1_053101.
• Fast 96-Well Plate, enter FastPureDye_Plate<plate #>_<date in
MMDDYY format>.sds
For example, for a fast pure dye plate 1 run on May 31, 2001, type:
FastPureDye_Plate1_053101.sds
• Microfluidic pure dye card, enter <Dye><date in MMDDYY format>_MFC
For example, the file name for a run performed on May 31, 2001 would be:
FAM_053101_MFC.
d. Click
.
The software saves the plate document.
7. Prepare and run the pure dye plate or card as explained below.
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Chapter 7 Maintaining the Instrument
Running the Prepared Pure Dye Plate or Card
Running the
Pure Dye Plate or
Card
1. Load the pure dye plate or microfluidic pure dye card into the 7900HT
instrument:
a. In the plate document in the SDS software, select the Instrument tab.
b. In the Real-Time tab in the Instrument tab, click
The instrument tray rotates to the OUT position.
.
c. Place the pure dye plate or card into the instrument tray as shown below.
Well A1
Position the plate so that the bar code
faces towards the front of the instrument
Well A1
Position the microfluidic pure dye card so that the
bar code faces towards the front of the instrument
Note: The A1 position is located in the top-left side of the instrument tray.
2. Click
.
The 7900HT instrument begins the pure dye run. The method for a pure dye run
is coded into the software and consists of a single 2-min hold at 60 °C.
Note: Before starting the run, the instrument may pause (up to 15 min) to heat
the heated cover to the appropriate temperature.
3. When the pure dye run is complete and the Run Complete dialog box appears:
a. Click
to close the dialog box.
b. Click
instrument tray.
, and remove the pure dye plate or card from the
c. Extract the pure dye calibration information as explained on page 7-25.
7-24
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Performing a Pure Dye Run
Analyzing the Pure Dye Run
Extracting Pure
Dye Information
from the Run
The purpose of viewing the data in the Pure Dye Wizard is to eliminate irregular pure
dye peaks from the data set. The wizard presents the spectral data from the pure dye
plate or card in sets of three wells, each containing the same pure dye. Because the
wells displayed by the wizard contain the pure dye at an identical concentration, the
signal peaks for the set should be identical. Occasionally, pipetting inaccuracies or
contamination can cause a well signal to shift slightly. While viewing the data, the
outlying peaks must be eliminated.
1. Select Analysis > Extract Pure Dye Wizard.
2. Follow the instructions as explained by the Extract Pure Dye Wizard to extract
the pure dye spectra.
When presented with each screen, do the following:
a. Inspect the spectra for shifts in peak location.
Example Spectra from a Pure Dye Plate:
Wavelength
shift
Click here to
remove it
Example spectra from a Microfluidic Pure Dye Card:
Wavelength
shift
Click here to
remove it
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7-25
Chapter 7 Maintaining the Instrument
b. If the data set contains an outlying peak, eliminate it by clicking the check
box of the associated well.
Note: Dye spectra are generally acceptable if they peak at the same location as
their group but diverge slightly at other wavelengths.
c. Click
when finished.
d. Repeat steps a to c for all remaining wells until prompted with a message
reporting the extraction of the pure dyes.
The software extracts the pure spectra and stores the data as a component of the
calibration file.
3. Click
(or select File > Save).
The software saves the plate document.
4. Select File > Close.
The software closes the plate document.
5. If performing spectral calibration of a:
• Standard 384-Well Block – The pure dye calibration is complete.
• Standard 96-Well Block – Run the second Pure Dye plate by repeating the
procedures for:
– “Preparing a Plate Document for a Pure Dye Plate or Card” on page 7-22
– “Running the Prepared Pure Dye Plate or Card” on page 7-24
– “Analyzing the Pure Dye Run” on page 7-25
• 7900HT System TaqMan Low Density Array Upgrade – Run the second
and third microfluidic pure dye cards containing the VIC and ROX dyes by
repeating the procedures for:
– “Preparing a Plate Document for a Pure Dye Plate or Card” on page 7-22
– “Running the Prepared Pure Dye Plate or Card” on page 7-24
– “Analyzing the Pure Dye Run” on page 7-25
7-26
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Adding Custom Dyes to the Pure Dye Set
Adding Custom Dyes to the Pure Dye Set
When to Perform
Materials
Required
Creating a
Custom Pure Dye
Plate
The Applied Biosystems 7900HT Fast Real-Time PCR System can be used to run
assays designed with custom dyes (dyes not manufactured by Applied Biosystems).
However, before using custom dyes with the 7900HT instrument, you must create
and run a custom pure dye plate.
• ABI PRISM™ 384- or 96-Well Optical Reaction Plate or Optical 96-Well Fast
Thermal Cycling Plate
• An optical adhesive cover or ABI PRISM™ Optical Flat Caps
• Custom Dye(s)
• Pipettor, 100-µL (with pipet tips)
• Centrifuge, with plate adaptor
Note: The purpose of the custom pure dye plate is identical to that of an ABI PRISM
Pure Dye Plate. The SDS software uses the custom plate to create a spectral standard
for multicomponenting the custom dye.
1. Prepare a microplate with a dilution series of the custom dye.
2. Start the SDS software.
3. Create an allelic discrimination plate document and run the dilution series plate.
Note: It is not necessary to configure detector, sample, and method information for
the dilution series plate document. The purpose of the run is to establish the correct
working concentration for the dye by viewing the intensity of the raw spectra
produced by the wells in the dilution series.
4. Select Analysis > Analyze.
The software analyzes the raw run data.
5. Click
(Show Raw Data Plot).
6. In the Raw Data Plot, determine the highest concentration of dye that does not
produce a saturated signal, and record it for future use.
Note: Saturated signals are characterized by their high peaks that rise beyond
detectable levels (> 65,000 fluorescent units) and appear as plateaus on the
Raw Data plot.
The concentration of the custom dye that yields the highest possible signal but
does not saturate is the maximum concentration for use with the 7900HT
instrument.
7. Repeat steps 1 through 6 for any additional custom dyes.
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Chapter 7 Maintaining the Instrument
GR2107
Custom Dye 8
Custom Dye 7
Custom Dye 6
Custom Dye 5
Custom Dye 4
Custom Dye 3
Custom Dye 2
Custom Dye 1
8. Create a pure dye plate for the custom dye(s) by pipetting each custom dye to at
least three columns of an optical plate at the concentrations determined in
step 7.
IMPORTANT! The optical configuration of the 7900HT instrument requires that
each pure dye occupy at least three columns of the Pure Dye plate to permit
adequate data collection.
9. Seal the plate using an optical adhesive cover or optical flat caps.
10. Create a plate document template for the custom Pure Dye plate as explained
below.
Constructing a
Custom Pure Dye
Plate Document
Template
1. Start the SDS software.
2. Add the new dye to the software using the Dye Manager:
a. Select Tools > Dye Manager.
b. In the Dye Manager dialog box, click
.
c. In the Add Dye dialog box, enter a name for the custom dye, and
click
.
The software adds the new dye to the Custom dye list.
d. Repeat steps b and c to add any additional custom dyes to the
Dye Manager.
e. Click
.
The SDS software makes the new dyes available to pure dye plate documents.
3. Create a custom pure dye plate document for the run:
a. Click
(or select File > New).
b. Configure the New Document dialog box with the following options:
Assay – Select Pure Spectra.
Container – Select the appropriate plate format.
Template – Select Blank Template.
c. Click
7-28
. The software creates a new plate document.
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Adding Custom Dyes to the Pure Dye Set
4. Apply pure dyes to the custom plate document:
a. Select the wells containing the custom dye.
b. In the Dyes drop-down list in the Setup tab, select the appropriate dye.
The software applies the dye to the selected wells.
c. Repeat steps a and b to configure the plate document with any additional
custom dyes.
Dyes drop-down list
Custom dye added to selected
wells of the plate document
5. Save the custom Pure Dye plate document as a plate document template:
a. Click
(or select File > Save).
b. In the Save dialog box, navigate to Program Files > Applied
Biosystems > SDS 2.2.1 > Templates.
The Templates directory appears inside the Look in field. By saving the
plate document template to the Templates directory, it becomes available
from the Template drop-down list in the New Document dialog box.
c. Select Files of type > SDS 7900HT Template Document (*.sdt).
d. In the File name field, and enter a name for the plate document template.
e. Click
template.
. The software saves the plate document as a plate document
6. Run the custom Pure Dye plate as explained in “Preparing the Pure Dye Plates”
on page 7-22.
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Chapter 7 Maintaining the Instrument
Verifying Instrument Performance
When to Perform
Applied Biosystems recommends performing an RNase P or TGF-β run:
• When changing sample block formats for the first time
• As needed to verify the function of the 7900HT instrument
Purpose of
RNase P Runs
The TaqMan RNase P Instrument Verification Plates are experiments run to verify
the performance of the 7900HT instrument for use with optical plates of the same
format (384-Well, 96-Well, or 96-Well Fast). The TaqMan RNase P Instrument
Verification Plates are pre-loaded with the reagents necessary for the detection and
quantification of genomic copies of the human RNase P gene (a single-copy gene
encoding the RNase moiety of the RNase P enzyme). Each well contains pre-loaded
reaction mix (master mix, RNase P primers, and FAM-labeled probe) and template.
Table 7-2 illustrates the arrangement of standards and samples on each type of
TaqMan RNase P Instrument Verification Plate. As shown below, the TaqMan RNase
P Instrument Verification Plates consist of five columns of template standards (1250,
2500, 5000, 10,000, and 20,000 copies) and two unknown populations (5000 and
10,000 copies).
Table 7-2
Configurations of the TaqMan RNase P Instrument Verification Plates
TaqMan RNase P
Instrument Verification
Plate
Sample Configuration
Unknown 2
10000
GR2107
Unknown 1
5000
NTC
STD 1250
STD 2500
STD 5000
STD 10000
STD 20000
TaqMan® RNase P 384-Well
Instrument Verification
Plate (PN 4323306)
7-30
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Verifying Instrument Performance
Table 7-2
Configurations of the TaqMan RNase P Instrument Verification Plates
TaqMan RNase P
Instrument Verification
Plate
Sample Configuration
TaqMan® RNase P
Instrument Verification
Plate
(PN 4310982, standard
96-well plate)
Unknown 1
5000
NTC
STD 1250
STD 2500
STD 5000
STD 10000
STD 20000
GR2108
Unknown 2
10000
Purpose of
TGF-β Runs
Materials
Required
The TaqMan® Low Density 7900HT Installation Array (TGF-β card) is an
experiment run to verify the performance of the 7900HT instrument for use with
TaqMan Low Density Arrays. The TGF-β card is preloaded with the reagents
necessary for the detection and quantification of genomic copies of the TGF-β gene.
Each well contains preloaded TGF-β primers and FAM MGB dye-labeled probe.
RNase P Runs
• Appropriate TaqMan RNase P Instrument Verification Plate (see Table 7-2 on
page 7-30)
• Centrifuge, with plate adaptor
TGF-β Runs
•
•
•
•
•
•
TGF-β card
TaqMan® 2✕ Universal PCR Master Mix
Water, DNase/RNase-free, deionized
2-mL microtube, sterile
Centrifuge
Microfluidic card sealer
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7-31
Chapter 7 Maintaining the Instrument
Preparing the
Verification Plate
or TGF-β Card
Preparing a TaqMan RNase P Instrument Verification Plate
1. Remove the TaqMan RNase P Instrument Verification Plate from the freezer
and allow it to thaw to room temperature.
2. Briefly centrifuge the TaqMan RNase P Instrument Verification Plate.
3. Continue with “Preparing the Plate Document” on page 7-33.
Preparing a TGF-β Card
4. Remove the TGF-β card from the refrigerator and allow it to warm to room
temperature.
5. In a sterile 2-mL tube, prepare the following PCR reaction mixture:
Reagent
Volume (µL)
TaqMan 2✕ Universal PCR Master Mix
500
Human cDNA
50
RNase/DNase-free water
450
Total Volume
1000
6. Tap the tube gently to mix.
7. Pipette 100 µL of the reaction mix into each of the eight fill reservoirs.
8. Continue with “Preparing the Plate Document” on page 7-33.
7-32
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Verifying Instrument Performance
Preparing the
Plate Document
1. Start the SDS software.
2. Click
(or select File > New).
3. Configure the New Document dialog box.
Assay – Absolute Quantification
Container – Select the appropriate plate format.
Template – If running a:
• TaqMan® RNase P 384-Well Instrument Verification Plate, select
384 Well RNaseP Install Plate.sdt.
• TaqMan® RNase P Instrument Verification Plate (standard 96-well plate),
select 96 Well RNaseP Install Plate.sdt.
• TaqMan® RNase P Fast 96-Well Instrument Verification Plate, select
96 Well RNaseP Install Plate.sdt.
• TGF-β card, select rnase p card template.sdt.
Note: If no plate document templates are available, construct a Pure Dye plate
document using the product insert from the Spectral Calibration Kit and the
procedure on page 7-28.
4. If desired, enter the bar code information into the plate document:
a. Click the Barcode text field.
b. Remove the TaqMan RNase P Instrument Verification Plate or TGF-β card
from the packaging and scan its bar code using the hand-held bar code
reader.
5. Click
. The software creates a plate document.
Note: Do not modify the plate document. The plate document template is
pre-programmed with detector and method information for the run.
6. Save the plate document:
a. Click
(or select File > Save).
b. In the Barcode field of the Save dialog box do one of the following:
– Enter a name or bar code number for the plate, then click
.
– Using the hand-held bar code reader, scan the bar code number.
c. Select Files of type > SDS 7900HT Document (*.sds).
d. Click
.
The software saves the plate document. The software is now configured for the
RNase P run.
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7-33
Chapter 7 Maintaining the Instrument
Running the
Verification Plate
or TGF-β Card
1. In the plate document in the SDS software, select the Instrument tab.
2. In the lower portion of the Instrument tab, select the Real-Time tab.
3. If the instrument tray is inside the 7900HT instrument, click
.
The instrument tray rotates to the OUT position.
4. Place the TaqMan RNase P Instrument Verification Plate or TGF-β card into the
instrument tray.
Note: The A1 position is located in the top-left corner of the instrument tray.
5. In the Real-Time tab, click
. The 7900HT instrument begins the run.
Note: Before starting the PCR run, the instrument may pause (up to 15 min) to
heat the heated cover to the appropriate temperature.
6. When the run is complete:
a. Analyze the run data as explained on page 6-12.
b. Set the baseline and threshold values for the analyzed data as explained on
page 6-46.
c. Verify the performance of the 7900HT instrument as explained below.
Verifying
Instrument
Performance
Analyzing Data from RNase P Runs
The install specification of the Applied Biosystems 7900HT Fast Real-Time PCR
System demonstrates the ability to distinguish between 5,000 and 10,000 genome
equivalents with a 99.7% confidence level for a subsequent sample run in a single
well.
The following equation verifies the 7900HT instrument install specifications:
[ ( CopyUnk 1 ) – 3 ( σ CopyUnk1 ) ] > [ ( CopyUnk 2 ) – 3 ( σ CopyUnk2 ) ]
where:
• CopyUnk1 is the Average Copy Number of Unknown #1 (10,000 replicate
population)
• σCopyUnk1 is the Standard Deviation of Unknown #1 (10,000 replicate population)
• CopyUnk2 is the Average Copy Number of Unknown #2 (5000 replicate
population)
• σCopyUnk2 is the Standard Deviation of Unknown #2 (5000 replicate population)
Note: The values above can be obtained from the experimental report window.
Note: Up to six wells from each replicate group in a 96-well TaqMan RNase P
Instrument Verification Plate (both standard 96-well and Fast 96-well plates) can be
ignored to meet specifications.
Note: Up to 10 wells from each replicate group in a 384-well TaqMan RNase P
Instrument Verification Plate can be ignored to meet specifications.
7-34
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Section 7.2 Maintaining the Plate Handler
Section 7.2 Maintaining the Plate Handler
In This Section
Automation Accessory Components and Stack Positions. . . . . . . . . . . . . . . . . . . 7-36
Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . 7-37
Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41
Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49
Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52
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Chapter 7 Maintaining the Instrument
Automation Accessory Components and Stack Positions
Refer to Figure 7-5 for the components discussed in this section.
(cross-sectional view of the gripper)
GR2014a
Automation
Accessory
Components
Plate-sensor Gripper
switch
Adjustment
knob
Plate stack
Expansion stacks
Zymark Twister Microplate Handler
Fixed-position bar code reader
Figure 7-5
Plate Stack
Positions
Components of the Automation Accessory
The Zymark Twister Microplate Handler alignment is performed using the Zymark®
Twister Software. The software refers to the positions of the plate stacks differently
than the Automation Controller Software. Figure 7-6 lists the positions defined by
the Zymark Twister Software and the Automation Controller Software equivalents.
1
0
7
2
6
3
(front of instrument)
4
5
Bar code
Well A1
Figure 7-6
7-36
Zymark Twister
Software
Automation
Controller
Software
Position 0
Output
Position 1
(unused)
Position 2
Instrument
Position 3
(unused)
Position 4
Stack 1
Positions of the Automation Controller and Zymark Twister Software
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Adjusting the Sensitivity of the Plate Sensor Switch
Adjusting the Sensitivity of the Plate Sensor Switch
When to Perform
The plate sensor switch located underneath the arm of the Zymark Twister
Microplate Handler requires adjustment under the following circumstances:
• When changing sample block module formats
• If the Plate Handler is having difficulty sensing plates
Materials
Required
Adjusting the
Plate Sensor
Switch
• Optical plate (of the current sample block format)
The dimensions of different plate formats can place different requirements on how
the Plate Handler grips plates. To ensure smooth operation of the Automation
Accessory, adjust the plate sensor switch when changing consumable formats.
1. Power off the Zymark Twister Microplate Handler.
PHYSICAL HAZARD. The Zymark Twister Microplate
Handler must be powered off at all times during the following procedure. Failure
to comply can result in physical injury to the user or damage to the Plate
Handler.
2. Clear the switch position by turning the thumb wheel all the way to the Up
extreme (as indicated on the side panel).
GR2014a
Thumb wheel
Plate-sensor switch
3. Begin the adjustment of the sensor switch:
a. Grasp an optical plate by the sides, making sure not to place pressure in the
center of the plate to deform it.
b. Place the plate between the fingers of the gripper assembly and align it to
the middle of the centering device.
c. While holding the plate in position, slowly turn the thumb wheel to lower
the switch onto the reaction plate until the switch:
– Contacts the top of the plate, and
– Emits a soft, audible “clicking” noise
IMPORTANT! The sound emitted by the sensor switch is very faint and may be
difficult to hear. To make the adjustment easier, place your ear close to the
sensor switch while making the adjustment and listen for the switch to engage.
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Chapter 7 Maintaining the Instrument
4. Remove the plate and listen for the plate-sensor switch to disengage.
Did you hear the
switch disengage?
No
Then…
1. Move the switch Down a few steps by turning the thumb
wheel in the direction indicated on the arm.
2. Replace the plate inside the gripper and listen for the
switch to engage:
– If you do not hear the switch engage, then remove the
plate and repeat steps 1 and 2 above.
– If you hear the switch engage, remove the plate and
continue to step a below.
Yes
1. Move the switch Up by turning the thumb wheel one step
in the direction indicated on the arm.
2. Replace the plate and listen for the switch to engage:
– If you hear the switch engage, remove the plate and
repeat steps 1 and 2 above.
– If you do not hear the switch engage, then you have
successfully identified the zero point of the
plate-sensor switch.
Note: At the zero point, one step of the thumb wheel in the
Down direction causes the switch to engage.
5. After the zero point is established, carefully turn the thumb wheel in the Down
direction the number of steps appropriate for your plate format as indicated
below:
Consumable
Turn the thumb wheel in the Down direction…
ABI PRISM 96-Well Optical
Reaction Plate
20 steps
ABI PRISM 384-Well Optical
Reaction Plate
15 steps
TaqMan Low Density Array
15 steps
Note: If you lose count, begin again from step 4 and identify the zero point for
the switch.
6. Test the adjustment as explained on page 7-39.
7-38
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Adjusting the Sensitivity of the Plate Sensor Switch
Testing the
Adjustment
1. Place the reaction plate in the input stack 1 of the Plate Handler.
2. Power on the 7900HT instrument, the Plate Handler, and the computer.
3. Select
>
Programs >
Zymark Twister Plate Handler > Twister.
The Zymark Twister Software starts.
4. Click Manual Control.
5. In the Manual Control dialog box, click stack 4.
Click
The Plate Handler arm moves over the input stack.
6. Click
.
If the adjustment was successful, the Plate Handler arm will lower upon the
plate until the plate detector switch engages confirming the presence of the
plate.
If Plate Handler arm emits a grinding sound, adjust the plate sensor switch:
a. In the Zymark Twister Software, click
Handler arm.
to raise the Plate
b. Turn the thumbscrew in the Down direction 10 steps.
c. Repeat step 6 until the Plate Handler arm successfully detects the plate.
7. Click
, then click
.
If the adjustment was successful, the Plate Handler arm will grasp the plate and
remove it from the plate stack.
If Plate Handler arm stops before the gripper fingers are able to contact the plate
and fails to grasp or pick up the plate, adjust the plate sensor switch:
a. Turn the thumbscrew in the Up direction 10 steps.
b. Grasp the plate with one hand and, from the Zymark Twister Software,
click
to release the plate.
c. Replace the reaction plate into input stack 1 of the Plate Handler.
8. Repeat steps 6 through 7 until the Plate Handler arm successfully retrieves the
plate.
9. Grasp the plate with one hand and, from the Zymark Twister Software,
click
to release the plate.
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7-39
Chapter 7 Maintaining the Instrument
10. Exit the Zymark Twister Software.
a. Click
.
b. Click Quit Application to close the software.
A defect in the Zymark Twister Software can cause portions of the program to persist
in memory even after the software has been closed. Because the Zymark Twister
Software conflicts with the SDS software, the residual elements of the software must
be closed inside the Windows Task Manager before continuing.
1. Confirm that the stack has closed by viewing the Task Manager.
a. Press the Crtl + Alt + Del keys in unison.
b. In the Windows Security dialog box, click Task Manager.
c. In the Task Manager dialog box, confirm that the software has closed by
looking for the Zymark Twister Software entry in the Task list. If the
software is still running, click the software entry and click Close to exit the
remaining software.
d. Select File > Exit.
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Aligning the Plate Handler
Aligning the Plate Handler
When to Perform
Perform the following procedure if the Applied Biosystems 7900HT Fast Real-Time
PCR System is moved or the Zymark Twister Microplate Handler becomes
misaligned.
Symptoms that the Plate Handler is out of alignment include:
• Excessive downward movement of the Plate Handler arm (the arm grinds when
grasping or releasing plates)
• The Plate Handler arm collides with the plate stacks
• The Plate Handler arm releases plates above the bottom of the plate stacks
• Reaction plates tip or tilt when placed into the instrument tray by the arm
Preparing the
Instrument for the
Alignment
1. Remove the covers for the fixed-position bar code reader and the underlying
platform.
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-position bar code reader
and underlying platform covers
2. Loosen the three black thumbscrews on the platform connecting the 7900HT
instrument and the Plate Handler base.
Black thumbscrews
3. Move the instrument tray to the out position.
a. Start the Automation Controller Software.
If an error dialog box appears reading, ‘Machine calibration values are
not valid. Please refer to documentation for calibration process,’
click
.
b. Click
.
The 7900HT instrument moves the instrument tray to the out position,
perpendicular to the instrument.
c. Select File > Exit.
The software quits the Automation Controller Software.
4. Select
>
Programs >
Zymark Twister Plate Handler > Twister.
The Zymark Twister Software starts.
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Chapter 7 Maintaining the Instrument
5. Click Manual Control to display the Manual Control dialog box
Aligning
Input Stack 1
(Zymark
Position 4)
The alignment of input stack 1 (position 4 in the Zymark Twister Software) is the
first step in the alignment procedure. This alignment provides the basis for aligning
all subsequent stacks on the Plate Handler.
1. Place an empty plate into input stack 1(Zymark position 4).
2. In the Zymark Twister Software, click position 4.
Click
The Plate Handler arm moves over the input stack.
3. Using the Vertical Positioning commands, lower the Plate Handler arm until it
is just above the stack.
The Vertical Positioning box offers four ways to move the Plate Handler arm:
• Move the slider for large increments.
• Click inside the slider bar to move the arm in 250 step increments.
• Click the lower arrow on the bar to move the arm in 50 step increments.
• Click the up or down arrows in the Vertical Adjustment text box to move
the arm in 1-step increments.
• Click the Vertical Adjustment text box, enter a value, and press Enter to
move the arm into a specific location.
Slider
Slider bar (250 steps per click)
Down arrow (50 steps per click)
Text box arrows (1 step per click)
4. Check the rotary position of the Plate Handler arm to confirm that the gripper:
• is centered over the stack
• will not contact the sides of the stack when lowered
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Aligning the Plate Handler
5. Using the Rotary Adjustment arrows, adjust the rotational position of the
gripper so that it is centered over the input stack and will not contact the sides
when lowered.
• To move the Plate Handler arm clockwise, click the up arrow.
• To move the Plate Handler arm counter-clockwise, click the down arrow.
Up arrow (moves the arm clockwise)
Down arrow (moves the arm counter-clockwise)
6. Using the Vertical Positioning commands, carefully lower the Plate Handler
arm into the stack. Adjust the Rotary Adjustment value as needed to center the
gripper inside the stack.
7. After the gripper is centered inside the stack, click
.
The Plate Handler arm lowers upon the plate.
Confirm the following:
• the plate is in the middle of the gripper span
• the plate sensor switch is contacting the plate
• the gripper and plate do not contact the side of the stack
8. Click
.
The gripper grips the plate between its fingers.
9. Select
.
The Plate Handler raises the arm to its highest position. If the plate contacts the
sides of the stack, re-adjust the rotary position of the Plate Handler arm until the
plate moves freely in the stack.
Note: Contact between the plate and the stack or all stacks may be unavoidable.
However, try to minimize the contact as much as possible.
10. Using the Vertical Positioning commands, raise and lower the Plate Handler arm
several times to check the alignment.
11. Lower the Plate Handler arm to the bottom of the plate stack, click
and click
.
,
The software records the rotary position for the Zymark position 4
(input stack 1).
12. Click
.
The gripper releases the plate.
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Chapter 7 Maintaining the Instrument
Aligning the Plate
Handler to the
Instrument
The next step is to align the Plate Handler arm to the instrument tray (Zymark
position 2). This alignment will ensure a smooth exchange between the Plate Handler
arm and the instrument tray during operation of the instrument.
Note: The following procedure requires you to position the Plate Handler relative to
the 7900HT instrument, Before moving the Plate Handler, loosen the three black
thumbscrews on the platform connecting the 7900HT instrument and the Plate
Handler.
1. If not already present, place an empty plate into input stack 1 (Zymark
position 4) and pick it up with the Plate Handler arm:
a. In the Zymark Twister Software, click position 4.
b. Click
.
c. Click
.
2. Click position 2.
Click here
The Plate Handler arm moves over the instrument tray.
3. Use Vertical Positioning to lower the Plate Handler arm until it is approximately
1 cm above the instrument tray.
4. Using the Rotary Adjustment arrows, center the gripper and plate along the
Y-axis of the instrument tray.
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D
E
F
Center the plate
G
H
I
J
K
L
M
N
O
P
5. Center the gripper and plate along the X-axis of the instrument tray by sliding
the Plate Handler and base towards or away from the 7900HT instrument.
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
Center the plate
GR2012
P
6. Again, using the software to move the Plate Handler arm, center the gripper and
plate along the Y-axis of the instrument tray as explained in step 4.
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Aligning the Plate Handler
7. Using the Vertical Positioning commands, carefully lower the Plate Handler
arm onto the instrument tray and confirm that the plate rests squarely inside it.
8. Tighten the three black thumbscrews on the platform connecting the 7900HT
instrument and the Plate Handler.
9. Release the plate from the Plate Handler arm.
a. Click
.
b. Click
.
10. Click
.
The Plate Handler arm lowers onto the plate.
11. Save the rotary and vertical offset information:
a. Click
, and click
.
The software records the rotary position for the plate drawer (Zymark
position 2).
b. Click
, and click
.
The software records the vertical position for the plate drawer.
Re-checking the
Input Stack 1
Now that the positions of the Plate Handler and instrument are fixed, the Plate
Handler stacks can be aligned and the positional values recorded.
1. Place an empty plate into input stack 1.
2. In the Zymark Twister Software, click the icon for stack 4.
The Plate Handler arm moves over the input stack.
3. Using the Vertical Positioning commands, lower the Plate Handler arm until it
is 1 cm above the stack and verify that it is centered on the stack. If necessary,
center the stack using the Rotary Adjustment arrows.
4. Carefully lower the Plate Handler arm into the stack. Center the gripper as it
moves down the stack by adjusting the Rotary Adjustment arrows if needed.
5. After the Plate Handler arm is centered inside the stack, click
.
The Plate Handler arm lowers upon the plate.
Confirm the following:
• The plate is in the middle of the gripper span.
• The plate sensor switch is contacting the plate.
• The gripper does not contact the side of the stack.
6. Click
.
7. Click
.
The Plate Handler raises the arm to its highest position. If the plate contacts the
sides of the stack, re-adjust the rotary position of the Plate Handler arm until the
plate moves freely inside the stack.
Note: Contact between the plate and the stack or all stacks may be unavoidable.
However, try to minimize the contact as much as possible.
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Chapter 7 Maintaining the Instrument
8. Click
, and click
.
The software re-records the rotary position for the input stack 1 (Zymark
position 4).
9. While holding the plate, click
Defining the
Bottom of the
Stack
and remove the plate.
The Automation Controller Software requires a bottom position value for all stacks.
This value is used to prevent the Plate Handler arm from colliding or grinding as it
moves to the bottom of each stack.
1. Remove all plates from the instrument and the Plate Handler arm.
2. Place an empty plate into the output stack (Zymark position 0).
3. In the Zymark Twister Software, click position 0.
The Plate Handler arm moves over the output stack.
4. Using the Vertical Positioning commands, lower the Plate Handler arm until it
is just above the stack.
5. Check the rotary position of the Plate Handler arm to confirm that the gripper:
• is centered over the stack
• will not contact the sides of the stack when lowered
6. Using the Rotary Adjustment arrows, adjust the rotational position of the
gripper so that it is centered over the input stack and will not contact the sides
when lowered.
7. Using the Vertical Positioning commands, carefully lower the Plate Handler
arm into the stack. Adjust the Rotary Adjustment value as needed to center the
gripper inside the stack.
8. After the gripper is centered inside the stack, click
.
The Plate Handler arm lowers upon the plate.
Confirm the following:
• The plate is in the middle of the gripper span
• The plate sensor switch is contacting the plate
• The gripper does not contact the side of the stack
9. Click
.
The gripper grips the plate between its fingers.
10. Select
.
The Plate Handler raises the arm to its highest position. If the plate contacts the
sides of the stack, re-adjust the rotary position of the Plate Handler arm until the
plate moves freely in the stack.
Note: Contact between the plate and the stack may be unavoidable. However,
try to minimize the contact as much as possible.
11. Using the Vertical Positioning commands, raise and lower Plate Handler arm
several times to check the alignment.
12. Lower the Plate Handler arm and click
, and click
.
The software records the rotary position for position 0 (the output stack).
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Aligning the Plate Handler
13. Click the
.
14. While holding the plate, click
and remove the plate.
15. In the Vertical Adjustment field, enter –3200, and press Enter. The Plate
Handler lowers the arm to a position near the base of the output stack.
16. Carefully lower the Plate Handler arm until it is approximately 1–2 mm from the
bottom of the stack.
17. Click
, click
, and record the number in the Vertical
Adjustment field. The software records the vertical position for position 0 (the
output stack).
18. Click
position.
. The Plate Handler raises the Plate Handler arm to its highest
19. In the Vertical Adjustment field, enter the Vertical Offset value determined in
step 17, and press Enter. The Plate Handler lowers the Plate Handler arm to a
Vertical Offset position.
20. If necessary, readjust the Vertical Offset value and repeat steps 18 through 19
until satisfied with the setting.
Defining the
Positions of the
Remaining
Stacks
1. Place an empty plate into input stack 2 (Zymark position 5).
2. In the Zymark Twister Software, click position 5.
The Plate Handler arm moves over the input stack.
3. Using the Vertical Positioning commands, lower the Plate Handler arm until it
is approximately 1 cm above the stack and center it using the Rotary
Adjustment arrows.
4. Carefully lower the Plate Handler arm into the stack. Center the gripper as it
moves down the stack by adjusting the Rotary Adjustment arrows as needed.
5. After the Plate Handler arm is centered inside the stack, click
.
The Plate Handler arm lowers upon the plate.
Confirm the following:
• The plate is in the middle of the gripper span.
• The plate sensor switch is contacting the plate.
• The gripper does not contact the side of the stack.
6. Click
.
7. Click
.
The Plate Handler arm raises to its highest position. If the plate contacts the
sides of the stack, re-adjust the rotary position of the Plate Handler arm until the
plate moves freely in the stack.
Note: Contact between the plate and the stack may be unavoidable. However,
try to minimize the contact as much as possible.
8. Using the Vertical Positioning commands, raise and lower Plate Handler arm
several times to check the alignment.
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Chapter 7 Maintaining the Instrument
9. Click
, and click
.
The software records the rotary position for the Zymark position 5 (input
stack 2).
10. Repeat steps 1 through 9 for input stacks 3 and 4 to define Rotary Offset values
for the remaining positions 6 and 7:
Zymark position 7
(input stack 4)
Zymark position 6
(input stack 3)
11. Exit the Zymark Twister Software.
a. Click
.
b. Click Exit.
The software closes.
A bug in the Zymark Twister Software can cause portions of the program to
persist in memory even after the software has been closed. Because the Zymark
Twister Software conflicts with the SDS software, these residual elements must
be closed inside the Windows Task Manager before continuing.
12. Confirm that the Zymark Twister Software has closed by viewing the Task
Manager.
a. Press the Ctrl + Alt + Del keys in unison.
b. In the Windows Security dialog box, click Task Manager.
c. In the Task Manager dialog box, confirm that the Twister software has
closed by looking for the Twister software entry in the Task list. If the
software is still running, click the software entry and click End Task to
exit the software.
d. Select File > Exit to quit the Task Manager.
13. Replace the covers for the fixed-position bar code reader and the underlying
platform (removed in step 1 on page 7-41).
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-position bar code reader
and underlying platform covers
7-48
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Aligning the Fixed-Position Bar Code Reader
Aligning the Fixed-Position Bar Code Reader
Description
Preparing the
Instrument for the
Alignment
The fixed-position bar code reader must be set so that it automatically scans the
plate’s bar code as the plate is placed into the instrument tray by the Plate Handler.
1. Remove the cover for the fixed-position bar code reader.
GR2009
7900HT
Front view with Robot
7900HT FAST Real-Time PCR System
GR2009
Fixed-position bar code reader
cover
2. Power on the computer.
3. Start the Automation Controller Software.
4. Click
.
The 7900HT instrument moves the instrument tray to the out position,
perpendicular to the instrument.
5. Select File > Exit.
The software quits the Automation Controller Software.
Positioning the
Fixed-Position
Bar Code Reader
IMPORTANT! The instrument tray must be in the OUT position to align the bar code
reader.
1. Place a plate with bar code onto the instrument tray.
IMPORTANT! Orient the plate so that well A1 aligns to the A1 position of the
instrument tray and that the bar code faces the fixed-position bar code reader.
GR2018
Well position A1
Bar code
2. Select
>
Programs >
PSC Laser Data > LDHOST.
The LDHOST software starts and displays the LDHOST window.
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Chapter 7 Maintaining the Instrument
3. Establish communication with the fixed-position bar code reader:
a. Click
(Edit).
b. Click
(Terminal).
The software opens the Edit Configuration and Terminal dialog boxes.
c. In the Device Control dialog box, click
(Connect to Device).
The terminal window displays the fixed-position bar code reader response.
d. Click
to close the Information dialog box.
The LD Host program communicates with the bar code reader and updates the
Edit Configuration dialog box with the current configuration settings.
4. Configure the software for the alignment:
a. In the bottom of the Edit Configuration dialog box, locate and select the
Op. Modes tab.
Op. Modes tab
Note: You may need to use the arrows located in the bottom of the dialog box to
locate the Op. Modes tab.
b. In the Operating modes selection group of the Edit Configuration dialog
box, click the arrow to the right of the Mode heading and select Test from
the drop-down list.
Mode drop-down list
c. In the Device Control dialog box, click RAM to toggle to EEPROM
mode.
RAM button
d. In the Device Control dialog box, click Send.
e. In the Confirm dialog box, click YES to save to EEPROM.
The fixed-position bar code reader begins a continuous repeating scan of the bar
code. The software updates the Terminal dialog box every 0.5 sec indicating the
percentage of accurate reads completed during the 0.5 sec interval.
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Aligning the Fixed-Position Bar Code Reader
5. Loosen the black positional adjustment knob on the fixed-position bar code
reader, and position the scan head of the reader as far as possible from the plate
while maintaining the orientation towards the bar code on the plate (see below)
GR2018
Scan head of the fixed-position
bar code reader
Black positional
adjustment knob
6. While watching the Terminal dialog box, slowly adjust the orientation of the
fixed-position bar code reader until the percent successful reading displays the
highest number possible.
Percent successful reads
Note: It may be helpful to briefly place a sheet of white paper in front of the
plate bar code to view the area scanned by the laser.
7. When satisfied with the alignment, tighten the black positional adjustment knob
on the fixed-position bar code reader.
8. Restore the fixed-position bar code reader to normal operation:
a. In the Edit Configuration dialog box, change from Test back to Serial on
Line.
Mode drop-down list
b. In the Device Control dialog box, confirm that EEPROM is still selected,
and click Send.
c. In the New Decision dialog box, click YES to save to EEPROM.
The bar code reader stops scanning the plate bar code and resumes normal
operation.
9. Click Exit (
) to quit the LDHOST window.
The LDHOST window closes.
10. Replace the cover for the fixed-position bar code reader (from step 1 on
page 7-49).
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Chapter 7 Maintaining the Instrument
Cleaning and Replacing Gripper Finger Pads
When to Perform
Materials
Required
The adhesive used to affix bar code labels to certain brands of microplates can build
up on the gripper pads of the Zymark Twister Microplate Handler. Over time, the
residue can cause the gripper pads to stick to the microplates while handling them,
causing misfeeds. To prevent buildup, inspect the gripper pads monthly and clean or
replace the pads as needed.
Table 7-3
Materials Required for Replacing the Gripper Finger Pads
Material
Finger Pad Replacement Kit, containing 10 finger pads
Part Number
4315472
Flat-blade screwdriver, small
—
Phillips head screwdriver, small
—
Isopropanol in a squeeze bottle
—
CHEMICAL HAZARD. Isopropanol is a flammable liquid and
vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or
repeated contact may dry skin and cause irritation. It may cause central nervous
system effects such as drowsiness, dizziness, and headache, etc. Please read the
MSDS, and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
Cleaning the
Finger Pads
To clean the finger pads, wipe each pad thoroughly with Isopropanol until the residue
has been resolved. If the pads appear rough or the adhesive cannot be removed,
replace the pads as described below.
Replacing the
Finger Pads
1. Using the Phillips-head screwdriver, remove the two small Phillips-head screws
from the fingers on each side of the gripper, then remove the fingers.
Note: Move the Plate Handler arm into any position where it is easy to access
the screws.
2. Using a small flat-blade screwdriver, pry the worn finger pads off the fingers.
Note: The manufacturer recommends replacing all finger pads at the same
time.
3. Clean any residual adhesive off the fingers using isopropanol.
4. Remove a replacement finger pad from the paper backing, and place the finger
pad on the appropriate finger position.
5. Repeat for the remaining finger pads.
6. Install the fingers with the fingers pointing down and the finger pads toward the
center of the gripper.
7. Insert the screws into the fingers and tighten.
Note: The screws do not automatically align the grippers. Make sure that the
finger pads are making good contact with the plate when the arm grips a plate.
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Section 7.3 Maintaining the Computer and Software
Section 7.3 Maintaining the Computer and Software
In This Section
This section contains the following information:
General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54
Maintaining the SDS software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55
Note: The SDS software is a multicomponent system that must be maintained to
ensure optimal operation of the Applied Biosystems 7900HT Fast Real-Time PCR
System. Although an Applied Biosystems service engineer will complete most of the
maintenance, this section discusses important issues that you should understand.
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Chapter 7 Maintaining the Instrument
General Computer Maintenance
Maintenance
Schedule
The computer connected to the 7900HT instrument requires regular maintenance to
ensure reliable operation of the Applied Biosystems 7900HT Fast Real-Time PCR
System components. Applied Biosystems recommends the following tasks as part of
routine maintenance of the computer system:
Table 7-4
Maintenance Schedule for the Instrument Computer
Maintenance Task
Perform
Archive or Remove Old SDS Files
Weekly
Defragmenting the Hard Drive
Monthly or before
fragmentation reaches 10%
Upgrading the Operating System Software
When available/advisable
Upgrading the SDS Software
When available
Developing a Data Management Strategy
Applied Biosystems recommends developing a strategy for dealing with the files
produced by the SDS software. During a single day of real-time operation, the
Applied Biosystems 7900HT Fast Real-Time PCR System can generate over 200 MB
of data. Without a strategy for distributing and archiving SDS-related files, the
7900HT instrument can easily fill the hard drive of the computer within just a few
weeks of operation. See “Managing Sequence Detection System Data” on page 1-17
for a discussion of management strategies.
Archiving
SDS Files
To conserve space on the computer hard drive, SDS files can be archived using a data
compression utility. The compression utility archives files by encoding them in a
compressed form, thereby reducing the size of a file. SDS files can be compressed
and decompressed many times.
Several commercially available compression utilities are available. PKZIP and *.arc
are archive formats common to the Microsoft Windows operating system.
Defragmenting
the Hard Drive
Applied Biosystems recommends defragmenting the hard drive of the computer
attached to the instrument at least once every week or before fragmentation reaches
10%. As the Applied Biosystems 7900HT Fast Real-Time PCR System is used and
files are deleted and created, the free space on the computer hard drive eventually is
split into increasingly smaller blocks (called “clusters”). Consequently, as the SDS
software creates new files and extends old ones, the computer cannot store each file
in a single block. Instead, the system will ‘fragment’ the files by scattering their
component pieces across different sectors of the hard drive.
The fragmentation of SDS files decreases the performance of both the SDS software
and the computer operating system. As the hard drive becomes fragmented, programs
take greater time to access files because they must perform multiple seek operations
to access the fragments.
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Maintaining the SDS software
Several commercially available software utilities are available for repairing
fragmented file systems. The software utility defragments broken files by combining
their component pieces at a single location on the hard drive, thereby optimizing
system performance.
Upgrading the
Operating
System Software
Do not upgrade the operating system of the computer connected to the 7900HT
instrument unless instructed to do otherwise by an Applied Biosystems
representative. New versions of the Microsoft Windows operating system can be
incompatible with the SDS software and render it and the instrument inoperable.
Applied Biosystems service engineers maintain the operating system software as part
of planned maintenance visits. During the visit, an engineer will update the computer
operating system as upgrades become available and are validated by Applied
Biosystems.
Maintaining the SDS software
User Access
Requirement
Upgrading the
SDS Software
IMPORTANT! You must have administrator privileges on the computer to install
and/or upgrade the SDS software.
Applied Biosystems continually develops the SDS software to provide increased
functionality and reliability of the Applied Biosystems 7900HT Fast Real-Time PCR
System. As updates become available, Applied Biosystems sends notifications of the
upgrades to all Applied Biosystems 7900HT Fast Real-Time PCR System customers.
If an upgrade is user-installable, it can be found on the Applied Biosystems Company
Web site (see “How to Obtain Services and Support” on page xii).
Note: Applied Biosystems service engineers perform regular software updates
during planned maintenance visits.
Reinstalling the
Software
On rare occasions, when a piece of the SDS software becomes corrupt, it may be
necessary to re-install the software. In the event that the software must be
re-installed, observe the following guidelines to re-install or upgrade the software.
• Unless instructed to do otherwise, remove the SDS software using the uninstall
utility. Do not delete the program subdirectory from the Program Files directory.
• Install the SDS software under a user login that has administrator privileges on
the computer.
• Unless instructed to do otherwise, re-install the SDS software to the same
directory as the previous installation.
• Review all documentation accompanying the new software (such as installation
notes or user bulletin). The updated version of the software may contain new
features that require special consideration.
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Chapter 7 Maintaining the Instrument
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Troubleshooting
In This Chapter
8
8
Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2
Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5
Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9
Pure Dye Runs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11
Real-Time Runs (Quantitative PCR and Dissociation Curves). . . . . . . . . . . . . . . 8-12
End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13
Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14
Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . 8-17
TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18
SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20
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8-1
Chapter 8 Troubleshooting
Troubleshooting Table
Overview
The following table is designed to help you troubleshoot most of the problems you
may encounter while using the Applied Biosystems 7900HT Fast Real-Time PCR
System.
The information in the table is arranged by category as follows:
• Chemistry problems
• Run problems
• Instrument and Automation Accessory Problems
Each category contains subcategories, followed by a brief description of the
symptoms you might encounter.
To use this table, look for the category and the symptom you are experiencing. The
page number in the right-hand column corresponds to a description of the possible
cause(s) and recommended action(s) for that particular problem.
Table 8-1
Troubleshooting Table
Category
Symptom
Page
Chemistry and Run Problems
Chemistry
Low Precision
8-5
Irreproducibility
Software Installation
SDS Software
Unable to finish installation
(Install program appears to b e frozen)
8-5
Installation Interrupted
Run Problems
Background Runs
Software will not extract background data
8-9
Background is too high (greater than 2500)
Pure Dye Runs
Software will not extract pure dye data
8-11
Raw data from pure dye run appears strange
Signals plateau (saturation)
Signal is too low (< 10,000 FSU)
More than two outliers per dye in a single row
8-2
Real-Time Runs (Quantitative PCR and Dissociation Curves)
8-12
End-Point Runs (Allelic Discrimination)
8-13
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Troubleshooting Table
Table 8-1
Troubleshooting Table
Category
Symptom
Page
Instrument and Automation Accessory Problems
Software and 7900HT
Instrument
SDS software will not start
8-14
Software crashes/freezes the computer or
displays an error message
Communication error
Thermal cycler errors
Automation Controller Software cannot find a
plate document file
Computer and/or software displays the Run
Completed Successfully dialog box but will not
respond and appears to be frozen
Run will not start
Computer is slow when analyzing data,
opening or closing dialog boxes, and other
software processes.
The computer will not logon to the Windows
Operating System.
The computer will not boot up at all.
Zymark Twister Microplate
Handler and
Fixed-Position Bar Code
Reader
Plate Handler emits grinding noise when
picking up or putting down plates
8-17
Plate Handler arm contacts racks when
retrieving or stacking plates
Plate Handler arm releases plates awkwardly
into the plate stack
Reaction plates tip or tilt when placed into the
instrument tray by the Plate Handler arm
Plate Handler fails to sense or grasp plates
Plates stick to the gripper fingers of the Plate
Handler arm
Plate Handler does not restack plates in
original locations
Fixed-position bar code reader not reading
plate bar codes
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Chapter 8 Troubleshooting
Table 8-1
Troubleshooting Table
Category
Symptom
TaqMan® Low Density
Array
After removing the Low Density Array from its
packaging, the fill consumable is damaged
(creased, bent, or folded).
Page
8-18
After removing the Low Density Array from its
packaging, the aluminum foil backing is
damaged (creased, bent, or folded).
After removing the Low Density Array from its
packaging, dust or other particulates settle on
the reaction wells (optical side of the Low
Density Array).
After removing the Low Density Array from its
packaging, water condenses on the reaction
wells (optical side of the Low Density Array).
After pipetting, too little PCR reaction mixture
has gone into the fill reservoir.
After pipetting, some of the PCR reaction
mixture leaks out of the vent port in the fill
reservoir.
The PCR reaction mixture leaks during PCR
cycling.
After a mean assay result and standard
deviation of the replicates are calculated, a
specific replicate appears to be an outlier.
SDS Enterprise Database
Archive or restore operation was terminated
prematurely
8-20
Cannot Connect to the Database
8-4
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Low Precision or Irreproducibility
Low Precision or Irreproducibility
Overview
There are many reasons why an assay run with the Applied Biosystems 7900HT Fast
Real-Time PCR System can have less than optimal precision. Factors that can affect
precision are described in detail below.
Improper Threshold Setting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Imprecise Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Non-Optimized Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Incomplete Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Splashing PCR Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Writing on the Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluorescent Contamination on the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Errors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contaminated Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Improper or Damaged Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Low Copy Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Use of Non-Applied Biosystems PCR Reagents. . . . . . . . . . . . . . . . . . . . . . . . . .
Improper
Threshold Setting
8-5
8-6
8-6
8-6
8-6
8-6
8-7
8-7
8-7
8-7
8-7
8-8
8-8
8-8
The key to high-precision quantitative PCR is accurate detection of the geometric
phase. The Applied Biosystems 7900HT Fast Real-Time PCR System typically
delivers sufficient sensitivity so that at least three cycles of the geometric phase are
visible, assuming reasonably optimized PCR conditions. The SDS software
calculates a fixed signal intensity, called a threshold, that each signal generated from
PCR amplification must reach before it is recognized by the software as actual
amplification. The calculated threshold is an approximation, and should be examined
and modified as needed.
Modifying the Threshold
In a real-time document of the SDS software, the threshold can be modified via the
Amplification plot view following analysis of the run data. See “Setting the Baseline
and Threshold Values” on page 6-46 for more information.
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8-5
Chapter 8 Troubleshooting
Imprecise
Pipetting
The calculated quantities of target nucleic acid are directly affected by how precisely
the template volumes are added to the reaction mixes. Other individually added
reagents are also affected by pipetting precision (such as, variable magnesium affects
amplification efficiency).
Using Master Mixes
For this reason, Applied Biosystems highly recommends using a master mix. All
common components to a set of reactions should be mixed together and then
dispensed to the wells of the plate. Sub-master mixes can be used to further improve
the precision of identical replicates. For example, instead of pipetting 5 µL of the
same template into four replicate wells, pipette 20 µL of the template into a
sub-master mix, then divide the sub-master mix into four equal parts for
amplification. When making each master mix, add 5–10% additional volume to
compensate for pipetting losses.
Using Pipettors
Pipetting precision is also improved by:
•
•
•
•
Calibrating and servicing the pipettors regularly
Pipetting larger volumes
Reducing the number of pipetting steps whenever possible
Increasing the consistency of the pipetting method
Consult the manufacturer about the correct method of dispensing liquid volumes
accurately from the pipettor. For example, some pipettors are designed to deliver the
designated volume at the first plunger stop, so ‘blowing out’ the residue may cause
error. Also, before using a new pipettor tip to serially dispense a master mix, wet the
tip once by drawing up some of the master mix and dispensing it back into the mix
again.
Non-Optimized
Chemistry
Chemistries that have not been optimized may be susceptible to inconsistencies. To
maximize precision and reaction efficiency, optimize the primer and probe
concentrations of each individual assay used. Refer to the TaqMan Universal PCR
Master Mix Protocol (PN 4304449) for specific information about optimizing probe
and primer concentrations for TaqMan-related chemistries.
Incomplete
Mixing
For maximum precision, the PCR master mix must be mixed to uniformity. After all
reaction components are added to master mix, it should be vortexed for 4–5 seconds
before aliquoting it to the wells of the plate. Any dilutions performed during the
assay should also be vortexed.
Air Bubbles
Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, the
reagents would be applied to the wells using a pipetting technique that does not form
air bubbles. However, if a plate does contain air bubbles, they can usually be removed
by swinging, tapping, or briefly centrifuging the reaction plate.
Splashing PCR
Reagents
8-6
If PCR reagents splash the undersides of the optical adhesive covers, the heat from
the lid may bake the liquid to the cover and may distort the signal. If splashing
occurs, briefly centrifuge the reaction plate to remove all traces of liquid from the
caps.
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Low Precision or Irreproducibility
Drops
Drops of reagents that cling to the sides of the wells may not contact the thermal
cycler sample block and consequently may not amplify. If the drop slides into the mix
during PCR, then the amplified products will become diluted and the result will be
less than replicate wells that did not have drops. Therefore, carefully monitor the
reaction plate as it is being transferred into the thermal cycler or 7900HT instrument.
If you observe any drops, take steps to remove them, such as centrifugation.
Writing on the
Reaction Plates
Do not write on any surface of the optical plates or the optical adhesive covers. The
fluorescent properties of the ink can potentially affect the fluorescence emission
from the plate and alter the results. Instead, note the contents of each well on a sheet
of paper, or on a printout of the sample setup.
Fluorescent
Contamination on
the Plates
Many compounds found in laboratories are fluorescent. If they come into contact
certain optical surfaces, such as the optical adhesive covers, the fluorescent results
may be affected. For example, it has been noted that the powder used to lubricate the
insides of plastic gloves often contains fluorescent compounds. Use only powder-free
gloves and do not needlessly touch the reaction plates or optical adhesive seals.
Errors
Human errors from time to time are inevitable, such as pipetting into the wrong well,
or making a dilution mistake.
Human error can be reduced in the following ways:
• Perform the assay in a systematic fashion. For example, the pattern of sample
positions should be simple (avoid putting gaps in the rows).
• When pipetting the master mix, look directly down into the reaction plate so that
you can verify the transfer of the solution.
• If adding a small-volume reagent, such as template, place the drop of liquid on
the side of the well. Briefly tap or centrifuge the plate afterwards to bring the
droplet down into the well.
• After all pipetting is complete, visually inspect all the wells to confirm the
presence of the reagent drops. Tapping or centrifuging the reaction plate will
cause all the drops to slide down into the wells simultaneously.
• When making serial dilutions, be sure to change the pipet tip after each dilution
step.
• Visually inspect the liquid volumes being pipetted to verify that the volume is
approximately correct. A common mistake is using the wrong pipettor volume
setting (such as setting 20 µL instead of 2.0 µL).
• Visually inspect the volumes of the completed reactions, looking for any wells
that have volumes that do not match those of the other wells.
Contaminated
Sample Block
Any material contaminating the sample block can affect the results. For example,
mineral oil reduces thermal transfer. Residue from writing on reaction plates darkens
the wells, absorbing light.
The sample blocks should be periodically inspected for cleanliness. Sample block
contamination can be visualized by running a background plate and inspecting the
resulting background signal for aberrant peaks above 2500 FSU (see page 7-16). See
page 7-14 for instructions on decontaminating the sample block.
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8-7
Chapter 8 Troubleshooting
Improper or
Damaged
Plastics
Only Applied Biosystems optical plates, optical adhesive covers, and optical flat caps
should be used with the Applied Biosystems 7900HT Fast Real-Time PCR System.
The plastics that comprise the optical parts undergo special testing for the absence of
fluorescent impurities. Optical plates are frosted to improve the degree and precision
of light reflection. Bent, creased, or damaged plastics may adversely affect the
transmission of fluorescent signal or prevent proper sealing of a well resulting in
evaporation, change in sample volume, and altered PCR chemistry. Make sure to use
the correct plastics and visually inspect each reaction plate before use.
Note: See Appendix C, “Kits, Reagents, and Consumables,” for a list of compatible
consumables and reagents.
Low Copy
Templates
When amplifying samples that contain very low quantities of nucleic acid (generally
less than 100 molecules), expect lowered precision due to the Poisson distribution
and biochemical effects related to binding probabilities. Low copy templates are also
more susceptible to losses due to non-specific adhesion to plastic wells, pipettor tips,
etc. The addition of carrier to the sample, such as yeast tRNA or glycogen, can help
prevent these losses, increasing the precision and sensitivity of the assay.
Use of
Non-Applied
Biosystems PCR
Reagents
The Applied Biosystems buffer contains an internal passive reference molecule
(ROX™ dye), which acts as a normalization factor for fluorescent emissions detected
in the samples.
8-8
IMPORTANT! Non-Applied Biosystems PCR buffers may not contain the ROX
passive reference dye. If running non-Applied Biosystems chemistry, be sure to set
the passive reference for your experiment as explained on “Setting the Passive
Reference” on page 3-16.
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Background Runs
Background Runs
Background
Troubleshooting
Table
Table 8-2
Troubleshooting Background Runs
Observation
Possible Cause
Recommended Action
Software will not extract
background data
During setup, the wrong
plate type was assigned to
the plate document
Run a new background
plate document with the
proper plate type setting.
Background is too high
(≥ 2500 FSUa)
See below.
Sample block
contamination
1. Construct and run a new
background plate.
Background plate
contamination
2. See “Isolating
Sample Block
Contamination” below.
Background is too high
(=>2500)
aFluorescent standard units – The measure of amplitude displayed along the Y-axis of the
Background Plot.
Isolating
Sample Block
Contamination
Signals exceeding 2500 FSU are considered outside the limit of normal background
fluorescence and indicates that the either the background plate or the sample block
module may be contaminated.
1. If not already open, open the plate document for the background run.
2. In the toolbar, click
(Hide/Show System Raw Data Pane).
The SDS software displays the raw data pane for the background run.
3. Select all wells in the plate document.
4. Inspect the raw background data for an aberrant spectral peak or peaks.
Wells producing raw spectra that exceed 2500 FSU are considered irregular and
could be contaminated. The following figure illustrates the raw data produced
by a run on a sample block module containing a contaminated well.
Possible contamination
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8-9
Chapter 8 Troubleshooting
5. Identify the location(s) of the contaminated well(s) on the sample block by
selecting increasingly smaller regions of the plate document (see below).
a. The raw data from
the selected wells
contains the peak.
The contaminated
well must be in
columns 7-12.
b. The raw data from
the selected wells
does not contain
the peak.
The contaminated
well must be in the
last four wells of
columns 7-12.
c. The raw data from
the selected wells
contains the peak.
The contaminated
well must be in the
last four wells of
columns 10-12.
d. The raw data from
the selected wells
contains the peak.
The contaminated
well must be in the
last two wells of
columns 10-12.
e. By selecting each
of the wells from
the last two wells
of columns 10-12,
the location of the
contaminated
well (G10) is
determined.
8-10
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Pure Dye Runs
6. Repeat step 4 until you identify the location of each contaminated well.
7. Decontaminate the sample block as explained in “Decontaminating the Sample
Block” on page 7-14.
8. Run a background plate to confirm that the contaminants have been removed.
If the contamination is present after running the background plate for a second
time, the background plate is likely to be the source of contamination.
Pure Dye Runs
Pure Dye
Troubleshooting
Table
Table 8-3
Troubleshooting Pure Dye Runs
Observation
Possible Cause
Recommended Action
Software will not extract
pure dye data
During plate setup, the
wrong plate type was
assigned to the plate
document
Create and run a new pure
dye plate document with the
proper plate type setting
A background plate was not
run before the pure dye
plate or card
Run a background plate,
then run the pure dye plate
or card again
Pure dye plate or card was
loaded backwards
1. Verify the pure dye
wavelengths are as
expected.
Raw data from pure dye
run appears strange
(see below)
2. Rerun the pure dye plate.
or card
Signals plateau (saturation)
Intensity is set too high/low
Call Applied Biosystems
Technical Support.
• Evaporation
• Contamination
Rerun the pure dye plate or
card. If the problem
persists, discard the pure
dye plate or card and run a
new one.
Signal is too low
(< 10,000 FSU)
More than two outliers per
dye in a single row
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8-11
Chapter 8 Troubleshooting
Real-Time Runs (Quantitative PCR and Dissociation Curves)
Troubleshooting
Analyzed Data
When faced with irregular data, you can use the SDS software to diagnose some
chemistry- and instrument-related problems. The following table contains a
summary of checks for verifying the integrity of your run data and to help you begin
troubleshooting potential problems.
Raw Data Plot
The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for
the selected wells during each cycle of the real-time PCR.
What to look for:
• Signal tightness and uniformity – Do the raw spectra signals from replicate
groups and controls exhibit similar spectral ‘profiles’? If not, the plate or
sample block could be contaminated.
• Characteristic signal shape – Do the samples peak at the expected
wavelengths? For example, samples containing only FAM™ dye-labeled
TaqMan® probes should not produce raw fluorescence in the wavelength of a
VIC® dye component. A signal present in wells that do not contain the dye could
indicate that the sample, master mix, or well contains contaminants.
• Characteristic signal growth – As you drag the bar through the PCR cycles, do
you observe growth as expected? Absent growth curves may indicate a pipetting
error (well lacks template).
• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation
can be an indication that a well contains too much template or fluorescent signal.
Multicomponent
Plot
The Multicomponent Plot displays a plot of normalized multicomponent data from a
single well of a real-time run. The plot displays the component dye signals that
contribute to the composite signal for the well.
What to look for:
• Correct dyes displayed – Does the plot display all dyes as expected? The
presence of an unexpected dye may be the result of an error in detector setup,
such as assigning the wrong reporter or quencher dye.
• ROX dye fluorescence level – Does the ROX dye signal fluoresce below the
reporter dyes? If not, the lack of reporter fluorescence may be caused by an
absence of probe in the well (a pipetting error).
• Background fluorescence – Do all dyes fluoresce above the background? The
Background signal is a measure of ambient fluorescence. If a dye fails to fluoresce
above the background, it is a strong indication that the well is missing probes
labeled with the dye (well does not contain probe, PCR master mix, or both).
• MSE Level – The MSE (mean squared error) is a mathematical representation
of how accurately the multicomponented data fits the raw data. The higher the
MSE value, the greater the deviation between the multicomponented data and
the raw data.
8-12
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End-Point Runs (Allelic Discrimination)
Amplification Plot
The Amplification plot displays data from real-time runs after signal normalization
and Multicomponent analysis. It contains the tools for setting the baseline and
threshold cycle (CT) values for the run.
What to look for:
• Correct baseline and threshold settings – Are the baseline and threshold
values set correctly?
Identify the components of the amplification curve and set the baseline so that
the amplification curve growth begins at a cycle number that is greater than the
highest baseline number.
IMPORTANT! Do not adjust the default baseline if the amplification curve
growth begins after cycle 15.
Identify the components of the amplification curve and set the threshold so that it is:
• Above the background
• Below the plateaued and linear regions
• Within in the geometric phase of the amplification curve
• Irregular amplification – Do all samples appear to have amplified normally? The
three phases of the amplification curve should be clearly visible in each signal.
• Outlying amplification – When the run data is viewed in the CT vs. Well
Position plot, do replicate wells amplify comparably? Wells producing CT
values that differ significantly from the average for the associated replicate
wells may be considered outliers.
If a plate produces non-uniformity between replicates, some samples on the plate
could have evaporated. Check the seal of the optical adhesive cover for leaks.
End-Point Runs (Allelic Discrimination)
Troubleshooting
Analyzed Data
When faced with irregular data, you can use the SDS software to diagnose some
chemistry- and instrument-related problems. The following table contains a
summary of checks for verifying the integrity of your run data and to help you begin
troubleshooting potential problems.
Raw Data
The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for
the selected wells during each cycle of the PCR.
What to look for...
• Signal tightness and uniformity – Do the raw spectra signals from replicate
groups and controls exhibit similar spectral ‘profiles’? If not, the plate or
sample block could be contaminated.
• Characteristic signal shape – Do the samples peak at the expected
wavelengths? For example, samples containing only FAM dye-labeled TaqMan
probes should not produce raw fluorescence in the peak wavelength of the VIC
dye component. A signal present in wells that do not contain the dye could
indicate that the sample, master mix, or well contains contaminants.
• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation
can be an indication that a well contains too much template or fluorescent signal.
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8-13
Chapter 8 Troubleshooting
Software and 7900HT Instrument
Troubleshooting
Software and
Computer
Problems
Table 8-4
Troubleshooting Software and Computer Problems
Observation
Possible Cause
Recommended Action
Unable to finish installation
(Install program appears to
be frozen)
Installer dialog box is
hidden from view
1. Press and hold Alt.
2. Press Tab until the Installer
icon is selected.
3. Release both keys.
The installer dialog box returns
to the front of the desktop.
SDS Software version 2.2
does not function after the
installation of SDS Software
version 2.2.1
SDS Software version
2.2 is automatically
removed during the
installation
Reinstall the SDS Software
version 2.2
SDS software will not start
• Incorrect start-up
sequence
• Corrupted software
• Computer
hardware failure
• Operating System
(OS) corruption
• Loose bar code
reader cable
Follow the solutions listed until
the symptom goes away.
The software crashes/freezes
the computer or displays an
error message
1
1. Power off the 7900HT
instrument.
2. Check cable connections.
3. Restart the computer and
logon to the computer.
4. Power on the 7900HT
instrument.
5. Start the SDS software.
2
6. Restart the computer and
logon to your computer.
7. Reinstall the SDS software.
8. Start the SDS software.
3
Contact Applied Biosystems
Service for OS problems or if
the computer will not boot up at
all. You may have to reload the
OS from the CDs.
4
Contact Dell for troubleshooting
the computer hardware.
8-14
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Software and 7900HT Instrument
Table 8-4
Troubleshooting Software and Computer Problems
Observation
Possible Cause
Recommended Action
Communication error
Cables are connected
incorrectly
Check cable connections and
COM port setup. See
“Instrument Connections” on
page 1-10.
Thermal cycler errors
Sample block module
not fully engaged
Reseat the sample block
module as explained
“Replacing the Sample Block”
on page 7-6.
Automation Controller
Software cannot find a plate
document file
File not in correct
location
Remove file entry from plate
queue and add the file to the
plate queue again.
Dialog box does not respond
to mouse clicks or key
strokes
Java Runtime Error
Click the close box of the dialog
box to close it.
Run will not start
No calibration file
Perform background and pure
dye runs.
No background data in
calibration file
(background run has
not been performed)
See “Performing a Background
Run” on page 7-16 and
“Performing a Pure Dye Run”
on page 7-20.
No pure dye data in
calibration file
(pure dye run has not
been performed)
Calibration file does
not contain pure dye
data for a dye used on
the plate document
Calibration file was
created on another
instrument
Disk drive containing
the plate document
has less than 50 MB of
free space
Check the capacity of the
destination drive. If less than
50 MB of free space remains,
remove or archive existing data
files (see page 7-54).
Heated cover cannot
reach running
temperature because
no plate loaded
Open the instrument tray and
check that the instrument
contains a plate.
Instrument tray
contains a plate
Output stack contains
a plate or plates
Remove all plates from the
output stack of the Plate
Handler before starting the
queue.
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8-15
Chapter 8 Troubleshooting
Table 8-4
Troubleshooting Software and Computer Problems
Observation
Possible Cause
Recommended Action
Computer is slow when
analyzing data, opening or
closing dialog boxes, and
other software processes
Hard drive is
fragmented
Defragment the hard drive as
explained on “Defragmenting
the Hard Drive” on page 7-54.
Hard drive is almost
full
Remove or archive existing
data files as explained on
“Archiving SDS Files” on
page 7-54.
Logon window does
not appear
Restart the computer and logon
to your computer.
You are not logged on
as the Administrator
1. Logoff of your computer.
After the above
solutions have been
tried, the problem is
still not fixed
Contact Dell for troubleshooting
the computer hardware or OS.
Cables are not
connected or are not
seated properly
Check the cables.
The computer will not logon
to the Windows Operating
System
The computer will not boot
up at all
2. Logon again as the
Administrator.
The boot disk is corrupted.
1. Boot directly off of the
Windows NT® Operating
System Installation CD.
2. Boot off of the emergency
disk.
3. Reload the Windows NT
Operating System from the
CD.
After the above
solution has been
tried, the problem is
still not fixed
8-16
DRAFT
Contact Dell for troubleshooting
the computer hardware.
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Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader
Zymark Twister Microplate Handler and Fixed-Position Bar
Code Reader
Automation
Accessory
Troubleshooting
Table
Table 8-5
Troubleshooting the Automation Accessory
Observation
Possible Cause
Recommended Action
Plate Handler emits
grinding noise when
picking up or putting down
plates
Vertical offset too low
Re-align the Plate Handler
as explained in “Aligning the
Plate Handler” on
page 7-41.
Plate Handler arm contacts
racks when retrieving or
stacking plates
Plate Handler rotary offset is
incorrect or vertical offset is
too low
Plate detector switch set
too high
The Plate Handler arm
releases plates awkwardly
into the plate racks
Reaction plates tip or tilt
when placed into the
instrument tray by the
Plate Handler arm
Plate Handler fails to sense
or grasp plates
Plate sensor switch not
adjusted properly
Adjust the plate sensor
switch as explained in
“Adjusting the Sensitivity of
the Plate Sensor Switch” on
page 7-37.
Gripper pads on the fingers
of the Plate Handler arm are
worn or dirty
Change the gripper pads as
explained in “Cleaning and
Replacing Gripper Finger
Pads” on page 7-52.
Plates stick to the gripper
fingers of the Plate Handler
arm
Gripper pads are worn or
dirty
Change the gripper pads as
explained in “Cleaning and
Replacing Gripper Finger
Pads” on page 7-52.
Plate Handler does not
restack plates in original
locations
Restack when finished
option not selected
Configure the Automation
Controller Software to
restack the plates as
explained in page 4-42.
Fixed-position bar code
reader not reading plate
bar codes
Bar code reader is
misaligned
Re-align the fixed-position
bar code reader as
explained in “Aligning the
Fixed-Position Bar Code
Reader” on page 7-49.
Bar code reader is broken
DRAFT
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8-17
Chapter 8 Troubleshooting
TaqMan Low Density Array
Low Density Array
Troubleshooting
Table
Table 8-6
Troubleshooting the TaqMan® Low Density Array
Observation
Possible Cause
Recommended Action
After removing the Low
Density Array from its
packaging, the fill
consumable is damaged
(creased, bent, or folded).
N/A
If the fill consumable is
damaged, then the PCR
reaction mixture might not
flow into the reaction wells
during centrifugation.
Discard the Low Density
Array.
After removing the Low
Density Array from its
packaging, the aluminum
foil backing is damaged
(creased, bent, or folded).
N/A
If the aluminum foil backing
is damaged, then the PCR
reaction mixture might not
flow into the reaction wells
during centrifugation.
Discard the Low Density
Array.
After removing the Low
Density Array from its
packaging, dust or other
particulates settle on the
reaction wells (optical side
of the Low Density Array).
N/A
Remove dust or particulates
by lightly tapping or blowing
on the reaction wells.
Room-temperature,
pressurized nitrogen or an
air blower may be used.
IMPORTANT! Be sure to
remove all dust and
particulates. The reaction
wells (optical side of the
Low Density Array) must be
free of dust and
particulates, (especially
fluorescent particulates)
After removing the Low
Density Array from its
packaging, water
condenses on the reaction
wells (optical side of the
Low Density Array).
The Low Density Array may
not have come to room
temperature before being
removed from its
packaging.
Remove condensation by
lightly blowing on the
reaction wells.
Room-temperature,
pressurized nitrogen or an
air blower may be used.
IMPORTANT! Be sure to
remove all water
condensation. The exterior
surface of the reaction wells
(optical side of the Low
Density Array) must be free
of water condensation.
8-18
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TaqMan Low Density Array
Table 8-6
Troubleshooting the TaqMan® Low Density Array
Observation
Possible Cause
Recommended Action
After pipetting, too little
PCR reaction mixture has
gone into the fill reservoir.
The PCR reaction mixture
was not correctly pipetted
into the fill reservoir.
Care must be taken to
correctly pipette the PCR
reaction mixture (100 µL)
into the fill reservoir.
After pipetting, some of the
PCR reaction mixture leaks
out of the vent port in the
fill reservoir.
1. Carefully follow the
procedures provided in
“Loading the TaqMan
Low Density Arrays” on
page 4-14.
2. After centrifuging, be
sure to note the fluid
levels in the fill reservoir
as described in step 6 on
page 4-18.
The PCR reaction mixture
leaks during PCR cycling.
After a mean assay result
and standard deviation of
the replicates are
calculated, a specific
replicate appears to be an
outlier.
The aluminum foil backing
may have been damaged
during the sealing step.
N/A
Discard the Low Density
Array.
Note: See “About the
Sealer” on page 4-14 for
proper sealing instructions.
Review the multicomponent
analysis display for that
replicate. Delete the outlier
and reanalyze.
DRAFT
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8-19
Chapter 8 Troubleshooting
SDS Enterprise Database
SDS Enterprise
Database
Troubleshooting
Table
Table 8-7
Troubleshooting the SDS Enterprise Database
Observation
Possible Cause
Recommended Action
Cannot Connect to the
Database
N/A
To test or reset the database
connection:
1. Start the SDS software.
2. Log into the database as
a user with
Administrative privileges
3. Test and reconfigure the
database connection as
described in
“Reconfiguring the
Database Connection”
on page A-23.
Error message:
java.sql.SQLException:
Io exception: End of
TNS data channel
8-20
DRAFT
Rare error when
archiving or restoring a
large data set
Reattempt the archive or
restoration task.
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, CH_Trouble.fm
Software Reference
In This Appendix
A
A
Importing Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2
Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4
Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16
Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16
Operating the SDS Software from a Command Line . . . . . . . . . . . . . . . . . . . . . A-18
Using the Search Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-20
Connecting SDS Software to the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22
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A-1
Appendix A Software Reference
Importing Plate Document Setup Table Files
About the Import
Function
The SDS software features the ability to import setup table information (detector,
detector task, marker, and sample name layouts) into a plate document from a
tab-delimited text file. The import feature is designed to be a timesaving device that
facilitates the exchange of setup information between other programs and the SDS
software. Instead of setting up plate documents individually, a third-party program
can be used to construct setup table files, which can then be imported into plate
documents for use.
To guarantee a successful incorporation of setup information from a text file to the
plate document, the file must:
• Be saved in a tab-delimited text format
• Conform to the setup table file formats described on page A-15
Creating and
Importing Setup
Table Data into a
Plate Document
Importation of setup table data into a plate document is accomplished in three major
steps.
Creating an Empty Setup Table File
The first step in the procedure is to export a setup table file from a blank plate
document.
Note: The blank setup table file can be created using a secondary application (such
as Microsoft Excel or a text editor) so long as it is saved in tab-delimited format and
is configured according to the file structure explained on page A-15.
1. Start the SDS software.
2. Click
(or select File > New).
3. Configure the New Document dialog box with the correct assay type and plate
format for your experiment, and click
.
4. Click
(or select File > Export).
5. In the Look In field of the Export dialog box, navigate to the directory you
would like to receive the exported file.
6. Select Export > Setup Table.
7. Select the All Wells radio button.
8. Select the SDS 2.2.1 radio button.
9. Click the File name text box, and enter a name for the file.
10. Click
.
The software exports the setup table data for the empty plate document as a
tab-delimited text file.
11. Configure the setup table file with plate document information (detector, task,
marker, and sample data) as explained on page A-3.
A-2
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Importing Plate Document Setup Table Files
Configuring the Setup Table File with Plate Document Information
The second step in the procedure is to import the setup table file into a secondary
application, configure it with sample and detector information, and then save the
completed setup table file in tab-delimited format.
1. Start the application that you want to use to edit the setup table file.
2. Import the setup table file from the previous procedure as tab-delimited text.
If using a spreadsheet application to edit the setup table file, the application
automatically parses the tab-delimited information into the cells of a
spreadsheet.
3. Configure the setup table file with sample and detector information according
to the file structure explained on page A-4.
4. Save the setup table file in tab-delimited format.
5. Import the completed setup table file into an empty plate document as explained
below.
Importing the Completed Setup Table File into a Plate Document
The final step in the procedure is to import the completed setup table tab-delimited
file into an empty plate document.
1. If the plate document created in “Creating an Empty Setup Table File” on
page A-2 is still open in the SDS software, continue to step 3. Otherwise, create
a plate document to receive the setup table data:
a. Start the SDS software.
b. Create or open a plate document to receive the information from the text
file.
2. Click
(or select File > Import).
3. In the Look In field of the Import dialog box, navigate to and select the
completed tab-delimited setup table file from step 4 in the previous procedure.
4. Click
.
The software imports the setup table information from the text file and
automatically configures the plate document plate grid and setup table with
detector, detector task, marker, and sample data.
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A-3
Appendix A Software Reference
Setup Table Files
About the Setup
Table File Format
The SDS software features the ability to import setup table information into a plate
document from a tab-delimited text file. To guarantee a successful importation of
setup table data, the imported file must follow the setup table format outlined in this
section.
Format Overview
The information in the setup table file must be ordered according to the following
convention:
1. File Version (see page A-5)
Plate Characteristics
2. Plate Size (see page A-5)
3. Plate ID (see page A-5)
Detector Definitions
4. Number of Detectors (see page A-6)
5. Detectors List Header (see page A-6)
6. Detectors List (see page A-7)
Marker Definitions
7. Number of Markers (Allelic Discrimination Only) (see page A-8)
8. Markers List Header (Allelic Discrimination Only) (see page A-8)
9. Markers List (Allelic Discrimination Only) (see page A-9)
Well-Detector Information
10. Well-Detector List Header (see page A-10)
11. Well-Detector Definition List (see page A-10)
Well-Marker Information
12. Well-Marker List Header (Allelic Discrimination Only) (see page A-12)
13. Well-Marker Definition List (Allelic Discrimination Only) (see page A-12)
Conventions
The following conventions are used in the rest of this section:
• courier – Text appearing in bolded courier font must be applied to a setup
table file exactly as appears in this document.
• italic – Text appearing in italic courier font must be substituted with custom
values when applied to a setup table file.
• [ required text ] – Text appearing between brackets is required
information in setup table files. All information inside the brackets must be
present in the setup table file for the SDS software to import it.
• { optional text } – Text appearing between braces is optional in setup
table files.
• <tab> – The tab character (the equivalent of pressing the Tab key)
• <cr> – The carriage-return character (the equivalent of pressing the Enter key)
IMPORTANT! To guarantee a successful importation of the setup table file into a
plate document, the file must contain all the elements described in the following
section and in the order that they appear in this document.
A-4
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Setup Table Files
1. File Version
Description
This line defines the version of SDS Assay Plate File format used to generate the document.
Note: Assay Plate Setup Files of version 4 and higher cannot be imported directly
into SDS 2.0 or SDS 2.1.
Format
Example
[ *** SDS Setup File Version <tab> version number <cr> ]
*** SDS Setup File Version 3
Plate Characteristics
2. Plate Size
Description
Format
Example
This line defines the number of wells in the plate modeled by the file (for example,
384 or 96).
[ *** Output Plate Size <tab> number of wells <cr> ]
*** Output Plate Size
384
3. Plate ID
Description
Format
Example
This line defines the ID of the Assay Plate. Normally this is a bar code that is printed
on the plate.
[ *** Output Plate ID <tab> plate id <cr> ]
*** Output Plate ID
384N75822034
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A-5
Appendix A Software Reference
Detector Definitions
Element numbers 4 to 6 define the detectors that appear in the well descriptions that
follow in a later section. The detector definition consists of three sections: the
declaration of the number of detectors, the detector list header, and the detector list.
4. Number of Detectors
Description
This line defines the total number of detectors on the plate.
Format
[ *** Number of Detectors <tab> number of detectors <cr> ]
Example
*** Number of Detectors
5
5. Detectors List Header
Description
This line contains the column headings for the Detector Definitions section of the
setup table file that make the file easier to edit using a program such as Microsoft
Excel.
Format
[ Detector <tab> Reporter <tab> Quencher <tab> Description <tab>
Comments <cr> ]
Example
Example Code A-1
Detector
A-6
Detectors List Header
Reporter
Quencher
Description
DRAFT
Comments
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Setup Table Files
6. Detectors List
Description
The detector list consists of one or more lines displaying the information for each
detector used on the plate document.
• The Detectors List section must contain one line (or definition) for each
detector present on the plate.
• The number of lines in the Detectors List section must be equal to the number
defined in the Number of Detectors section (see number 4 above).
• Leave blank the Quencher Dye entry for detectors created for the SYBR Green I
Dye or probes labeled with a non-fluorescent quencher.
Format (for a
single detector):
[ detector name <tab> reporter dye <tab> quencher dye <tab>
description <tab> comments <cr> ]
Examples:
Example Code A-2
Allelic discrimination setup table file
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
CYP 2C9*2.1
FAM
CYP 2C9*2.2
VIC
...
Example Code A-3
3
384
384N75822034
2
Quencher
Description
PDAR CYP 2C9*2 Al1
PDAR CYP 2C9*2 Al2
Comments
Example Probe
Example Probe
Absolute or relative quantification setup table file
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
GAPDH
VIC
SYBR Green
SYBR
RNase P
FAM
...
3
96
96JG729FF903
3
Quencher
TAMRA
Description
GAPDH Probe
SYBR Green I
RNase P Probe
Comments
Example Probe
Example Probe
Example Probe
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A-7
Appendix A Software Reference
Marker Definitions
Element numbers 7 to 9 define the markers that appear in the well descriptions that
follow in a later section. The marker definition consists of three sections: the
declaration of the number of markers, the marker list header, and the marker list.
7. Number of Markers (Allelic Discrimination Only)
IMPORTANT! Number of Markers information is required only in setup table files
created for allelic discrimination runs. This section does not appear in setup table
files created for absolute or relative quantification runs.
Description
Format
Example
This line defines the total number of markers on the plate.
[ *** Number of Markers <tab> number of markers <cr> ]
*** Number of Markers
2
8. Markers List Header (Allelic Discrimination Only)
IMPORTANT! Marker List Header information is required only in setup table files
created for allelic discrimination runs. This section does not appear in setup table
files created for absolute or relative quantification runs.
Description
Format
This line contains the column headings for the Marker Definitions section of the
setup table file that make the file easier to edit using a program such as Microsoft
Excel.
[ Marker <tab> AlleleX <tab> AlleleY <tab> Description <tab> Comments
<cr> ]
Example
Example Code A-4
Marker
A-8
AlleleX
AlleleY
Description
DRAFT
Comments
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Setup Table Files
9. Markers List (Allelic Discrimination Only)
IMPORTANT! Marker List information is required only in setup table files created
for allelic discrimination runs. This section does not appear in setup table files
created for absolute or relative quantification runs.
Description
The marker list consists of one or more lines displaying the information for each
marker used on the plate document.
• The Markers List section must contain one line (or definition) for each marker
present on the plate.
• The number of lines in the Markers List section must be equal to the number
defined in the Number of Markers section (see number 7 above).
• Leave the markers list line blank in setup files generated for relative and
absolute quantification. (Real-time plate documents do not use markers.)
Format (for a
single marker):
[ marker name <tab> alleleX <tab> alleleY <tab> description <tab>
comments <cr> ]
Examples:
Example Code A-5
Allelic discrimination setup table file
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
CYP 2C9*2.1
FAM
CYP 2C9*2.2
VIC
*** Number of Markers
Marker
AlleleX
CYP 2C9*2
CYP 2C9*2.2
...
Example Code A-6
3
384
384N75822034
2
Quencher
1
AlleleY
CYP 2C9*2.2
Description
PDAR CYP 2C9*2 Al1
PDAR CYP 2C9*2 Al2
Comments
Example Probe
Example Probe
Description
PDAR CYP 2C9*2
Comments
Example marker
Absolute or relative quantification setup table file
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
GAPDH
VIC
SYBR Green
SYBR
RNase P
FAM
3
96
96JG729FF903
3
Quencher
TAMRA
Description
GAPDH Probe
SYBR Green I
RNase P Probe
Comments
Example Probe
Example Probe
Example Probe
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A-9
Appendix A Software Reference
Well-Detector Information
Element numbers 10 and 11 define the contents of the wells on the plate in terms of
detectors. The well-marker information consists of two sections: the Well-Detector
List Header and the Well-Detector Definition List.
10. Well-Detector List Header
Description
This line contains the column headings for the well-marker information section of
the setup table file that make the file easier to edit using a program such as Microsoft
Excel.
Format
[ Well <tab> Sample Name <tab> Detector <tab> Task <tab> Quantity ] {
... <tab> Detector <tab> Task <tab> Quantity } [ <cr> ]
Example
Example Code A-7
Well
Sample Name
Detector
Task
Quantity
Detector
Task
Quantity
11. Well-Detector Definition List
Description
This section defines the contents of the plate wells. The setup table file must contain a
definition for each well used on the plate. Each well definition list consists of one string
of characters terminated by a <cr>. The definition consists of three main functional
divisions:
• Well number – The first tab-delimited text block defines the number of the well on
the plate. Well numbers start at 1 for well A-1 (upper-left corner of the plate) and
increases from left to right and from top to bottom. The wells must be listed in
order (1,2,3,…).
• Sample name – The second text block defines the name of the sample assigned to
the well.
• Detector assignments – The remaining tab-delimited text blocks for the well
definition define the detectors assigned to the well. Each detector is represented by
three text blocks that define the following information:
– The name of the detector
– The task assignment of the detector for the well (UNKN - Unknown, STND Standard, NTC - No Template Control)
– The quantity assignment of the detector for the well. (For wells containing
standards, assign the quantity for the standard sample in initial copy number. For
all other wells, assign the quantity value as 0.)
To assign more than one detector to a well, repeat the detector definition text blocks for
each detector. There is no limit to the number of detectors that can appear in a well.
IMPORTANT! All detectors that appear in this section must have been previously
defined in the Detector Definitions section (elements 4–6).
A-10
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Setup Table Files
Format
(for a single well):
[ Well number
Detector
Detector
Detector
<tab> SDS Sample Name <tab> Detector name <tab>
task <tab> Detector quantity ] { <tab> Detector name <tab>
task <tab> Detector quantity … <tab> Detector name <tab>
task <tab> Detector quantity } [ <cr> ]
Example
Example Code A-8
Allelic discrimination setup table files
*** SDS Setup File Version
3
*** Output Plate Size
384
*** Output Plate ID
384N75822034
*** Number of Detectors
2
Detector
Reporter
Quencher
CYP 2C9*2.1
FAM
CYP 2C9*2.2
VIC
*** Number of Markers
1
Marker
AlleleX
AlleleY
CYP 2C9*2
CYP 2C9*2.2
CYP 2C9*2.2
Well
Sample Name
Detector
Task
1
Sample 1
2
Sample 2
3
...
384
Sample 48
Example Code A-9
Comments
Example Probe
Example Probe
Description
Comments
PDAR CYP 2C9*2
Example marker
Quantity
Detector
Task
Quantity
Absolute quantification setup table files
*** SDS Setup File Version
3
*** Output Plate Size
96
*** Output Plate ID
96JG729FF903
*** Number of Detectors
1
Detector
Reporter
Quencher
RNase P
FAM
TAMRA
Well
Sample Name
Detector
Task
1
Sample 1
GAPDH
UNKN
2
Sample 2
GAPDH
STND
3
...
96
Sample 30
GAPDH
UNKN
Example Code A-10
Description
PDAR CYP 2C9*2 Al1
PDAR CYP 2C9*2 Al2
Description
RNase P Probe
Quantity
0
20000
Comments
Example Probe
0
Relative quantification setup table files
*** SDS Setup File Version
3
*** Output Plate Size
96
*** Output Plate ID
96FRE505SDA2
*** Number of Detectors
2
Detector
Reporter
Quencher
GAPDH
VIC
TNF-α
FAM
Well
Sample Name
Detector
Task
1
Calibrator
GAPDH
ENDO
2
Sample 1
GAPDH
ENDO
3
...
96
Sample 12
GAPDH
ENDO
Description
GAPDH Probe
TNF-α Probe
Quantity
0
0
Comments
Example Probe
Example Probe
Detector
Task
TNF-α
UNKN
TNF-α
UNKN
Quantity
0
0
0
TNF-α
0
UNKN
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A-11
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Appendix A Software Reference
Well-Marker Information
Element numbers 12 and 13 define the contents of the wells on the plate in terms of
markers. The well-marker information consists of two sections: the Well-Marker List
Header and the Well-Marker Definition List.
12. Well-Marker List Header (Allelic Discrimination Only)
IMPORTANT! Well-Marker List Header information is required only in setup table
files created for allelic discrimination runs. This section does not appear in setup
table files created for absolute or relative quantification runs.
Description
This line contains the column headings for the well-marker information section of the
setup table file that make the file easier to edit using a program such as Microsoft Excel.
Format
[ Well <tab> Sample Name <tab> Detector <tab> Task <tab> Quantity ] {
... <tab> Detector <tab> Task <tab> Quantity } [ <cr> ]
Example
Example Code A-11
Well
Sample Name
Marker
Task
Marker
Task
13. Well-Marker Definition List (Allelic Discrimination Only)
IMPORTANT! Well-Marker Definition List information is required only in setup
table files created for allelic discrimination runs. This section does not appear in
setup table files created for absolute or relative quantification runs.
Description
This section defines the contents of the plate wells. The setup table file must contain a
definition for each well used on the plate. Each well definition list consists of one string
of characters terminated by a <cr>. The definition consists of three main divisions:
• Well number – The first tab-delimited text block defines the number of the well on
the plate. Well numbers start at 1 for well A-1 (upper-left corner of the plate) and
increases from left to right and from top to bottom. The wells must be listed in
order (1,2,3,…).
• Sample name – The second text block defines the name of the sample assigned to
the well.
• Marker assignments – The remaining tab-delimited text blocks for the well
definition define the marker assigned to the well. Each marker is represented by
two text blocks that define the following information:
– The name of the marker
– The task assignment of the marker for the well (UNKN - Unknown, NTC - No
Template Control)
To assign more than one marker to a well, repeat the marker definition text blocks for
each detector. There is no limit to the number of markers that can appear in a well.
IMPORTANT! All markers that appear in this section must have been previously
defined in the Marker Definitions section (elements 7–9).
A-12
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Setup Table Files
Format
(for a single well):
[ Well number <tab> SDS Sample Name <tab> Marker name <tab> Marker task ]
{ <tab> Marker name <tab> Marker task <tab> … } [ <cr> ]
Example
Example Code A-12
Allelic discrimination setup table files
*** SDS Setup File Version
3
*** Output Plate Size
384
*** Output Plate ID
384N75822034
*** Number of Detectors
2
Detector
Reporter
Quencher
CYP 2C9*2.1
FAM
CYP 2C9*2.2
VIC
*** Number of Markers
1
Marker
AlleleX
AlleleY
CYP 2C9*2
CYP 2C9*2.2
CYP 2C9*2.2
Well
Sample Name
Detector
Task
1
Sample 1
2
Sample 2
3
...
384
Sample 48
Well
Sample Name
Marker
Task
1
Sample 1
CYP 2C9*2
UNKN
2
Sample 2
CYP 2C9*2
UNKN
3
...
384
Sample 48
CYP 2C9*2
UNKN
Example Code A-13
Comments
Example Probe
Example Probe
Description
PDAR CYP 2C9*2
Quantity
Comments
Example marker
Marker
Task
Absolute quantification setup table files
*** SDS Setup File Version
3
*** Output Plate Size
96
*** Output Plate ID
96JG729FF903
*** Number of Detectors
1
Detector
Reporter
Quencher
RNase P
FAM
TAMRA
Well
Sample Name
Detector
Task
1
Sample 1
GAPDH
UNKN
2
Sample 2
GAPDH
STND
3
...
96
Sample 30
GAPDH
UNKN
Example Code A-14
Description
PDAR CYP 2C9*2 Al1
PDAR CYP 2C9*2 Al2
Description
RNase P Probe
Quantity
0
20000
Comments
Example Probe
Detector
Task
Quantity
Description
GAPDH Probe
TNF-α Probe
Quantity
0
0
Comments
Example Probe
Example Probe
Detector
Task
TNF-α
UNKN
TNF-α
UNKN
Quantity
0
0
0
TNF-α
0
0
Relative quantification setup table files
*** SDS Setup File Version
3
*** Output Plate Size
96
*** Output Plate ID
96FRE505SDA2
*** Number of Detectors
2
Detector
Reporter
Quencher
GAPDH
VIC
TNF-α
FAM
Well
Sample Name
Detector
Task
1
Calibrator
GAPDH
ENDO
2
Sample 1
GAPDH
ENDO
3
...
96
Sample 12
GAPDH
ENDO
UNKN
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Appendix A Software Reference
Example Setup Table Files
Example Code A-15
Relative quantification setup table document
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
18S-VIC-mgb VIC
Bcl-2
FAM
Bcl-xL
FAM
GAPDH
FAM
GBP2
FAM
GOS2
FAM
IL-15
FAM
IL-17
FAM
IL-18
FAM
IL-1a
FAM
IL-1b
FAM
IL12p40
FAM
INF-a2
FAM
IP-10
FAM
ISG56
FAM
Nfkb-I
FAM
PKCi
FAM
RAP1A
FAM
RhoPK2
FAM
TNF-a
FAM
c-fos
FAM
c-jun
FAM
c-myc
FAM
cot
FAM
p53
FAM
Well
Sample Name
1
A1
2
Liver
3
Liver
4
Liver
5
Liver
6
Liver
7
Liver
8
Liver
9
Liver
10
Liver
11
Liver
12
Liver
13
Liver
14
Liver
15
Liver
16
Liver
...
381
Liver
382
Liver
383
Liver
384
Liver
3
384
384JQZ55A4
25
Quencher
Description
Comments
Detector
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
Task
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
ENDO
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Human Bcl-2
Human Bcl-xL
Human GAPDH
guanlylate binding protein 2
Human GOS2 protein gene
Human IL-15
Human IL-17
Human IL-18
Human IL-1a
Human IL-1 beta
Human IL-12p40
Interferon-alpha2
Human IP-10
Interferon-induced protein 56
Nuclear factor of kappa light...
Protein kinase C, iota
RAP1A, member of RAS oncogene...
Homo sapiens Rho-associated...
Human TNF-alpha
Human c-fos
Human c-jun
Human c-myc
Cancer Osaka thyroid oncogene
Human p53
Quantity
0.0
GOS2
TARG
0.0
cot
TARG
0.0
cot
TARG
0.0
ISG56 TARG
0.0
ISG56 TARG
0.0
GOS2
TARG
0.0
cot
TARG
0.0
cot
TARG
0.0
ISG56 TARG
0.0
ISG56 TARG
0.0
GOS2
TARG
0.0
cot
TARG
0.0
cot
TARG
0.0
ISG56 TARG
0.0
ISG56 TARG
0.0
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
18S-VIC-mgb
ENDO
ENDO
ENDO
ENDO
0.0
0.0
0.0
0.0
GOS2
cot
cot
ISG56
TARG
TARG
TARG
TARG
0.0
0.0
0.0
0.0
Information for wells 17 through 380 has been removed from this example for brevity.
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Setup Table Files
Example Code A-16
Allelic discrimination setup table document
*** SDS Setup File Version
*** Output Plate Size
*** Output Plate ID
*** Number of Detectors
Detector
Reporter
AL2-1
FAM
AL1-1
VIC
AL2-2
FAM
AL1-2
VIC
AL2-3
FAM
AL1-3
VIC
AL2-4
FAM
AL1-4
VIC
*** Number of Markers
Marker
AlleleX
SNP1
AL2-1
SNP4
AL2-4
SNP3
AL2-3
SNP2
AL2-2
Well
Sample Name
1
A1
2
A2
3
A3
4
A4
5
A5
6
A6
7
A7
8
A8
9
A9
10
A10
11
A11
12
A12
13
A13
...
382
P22
383
P23
384
P24
Well
Sample Name
1
A1
2
A2
3
A3
4
A4
5
A5
6
A6
7
A7
8
A8
9
A9
10
A10
11
A11
12
A12
13
A13
...
382
P22
383
P23
384
P24
3
384
NU03000081
8
Quencher
4
AlleleY
AL1-1
AL1-4
AL1-3
AL1-2
Detector
Description
Comments
Description
Comments
Task
Quantity
Marker
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
SNP1
Task
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
SNP4
SNP4
SNP4
UNKN
UNKN
UNKN
Information for wells 11 through 381 has been removed from this example for brevity.
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Appendix A Software Reference
Exporting Graphics
Exporting a Plot
as a JPEG
Graphic File
The SDS software can export most panes and plots of the plate document as JPEG
(Joint Photographic Experts Group) graphic files. The JPEG file format is
compatible with most word processing and spreadsheet applications and can be
incorporated directly into HTML documents for viewing by most web browser
software.
To export an element of a plate document as a graphic:
1. Select the plot or grid you want to export.
2. Choose from the following:
– If exporting a plot, adjust its dimensions (length and width) as you want them
to appear in the exported graphic file. The exported graphic file retains the
dimensions of the original screen element.
– If exporting the plate grid, do not adjust the size of the wells. The software
captures the whole grid regardless of the size of the view.
3. Right-click the plot or grid, and select Save Plot/Grid to Image File from the
contextual menu.
Note: If a pane cannot be exported as a graphic, the contextual menu will not
contain the Save Plot/Grid to Image File option.
4. In the Save As dialog box, navigate to the directory you want to receive the
exported graphic file.
5. In the File name field, and enter a name for the new file.
6. Click
.
The software saves the plot or grid as a JPEG graphic in the designated
directory.
Exporting Plate Document Data
Exporting Data
from a Plate
Document
The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for
all or a select group of wells on a plate document. The exported files are compatible
with most spreadsheet applications.
To export an element of a plate document:
1. Click the plate document to select it.
IMPORTANT! The plate document must be the top-most object in the workspace.
2. Click
(or select File > Export).
3. Navigate to the directory you would like to receive the exported file(s).
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Exporting Plate Document Data
4. In the Export drop-down list, select the type of data you would like to export.
• Background Spectra – The exported file contains the fluorescence readings
for each well from the background component used to analyze the run
• Clipped – The exported file contains the average Rn and ∆Rn of the last
three data points collected during the extension phase of each cycle repetition
for each well
• Dissociation Curve – The exported file contains:
Temperature Data – For each well in use on the plate, the file displays the
calculated temperature of the wells during each data collection reading of the
temperature ramp.
Raw Data – For each well in use on the plate, the file displays the Rn of the
well during each data collection reading of the temperature ramp.
Derivative Data – For each well in use on the plate, the file displays the first
derivative data during each data collection reading of the temperature ramp.
• Multicomponent – The exported file contains:
– Amounts the calculated dye components in a single well throughout all
stages of the PCR that were labeled with data collection icons
– Pure spectra component data
– Calculated inverse matrix
– Singular values of the inverse matrix
• Pure Spectra – The exported file contains the fluorescence readings for each
well from the pure spectra calibration component used to analyze the run
• Raw Spectra – The exported file contains the unmodified fluorescence
readings taken for each spectral bin during the course of the run
• When exported, the software creates a directory and saves each the raw
spectra data for each well in a separate *.txt file.
• Results Table – The exported file contains the contents of the table pane of
an analyzed plate document
Note: The contents of the exported data vary depending on the type of plate
document.
• Setup Table – The exported file contains the contents of the table pane of a
plate document before analysis The contents of the exported data varies
depending on the type of plate document used to produce it. See page A-4 for
a detailed description of the Setup Table file.
5. To export data from:
• All wells of the plate document – Select the All Wells radio button.
• Selected wells of the plate grid – Select the Selected Wells radio button.
6. In the files of type drop-down list, select the appropriate format for the exported
data.
7. Click the File name text box, and enter a name for the exported file.
8. Click
.
The software saves the exported data to the designated location.
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Appendix A Software Reference
Operating the SDS Software from a Command Line
Overview
The SDS software allows you to use batch files to analyze and export results from the
command line. Use of the command line interface is intended for advanced users
(such as systems administrators, bioinformaticians, and laboratory network
administrators) who choose to drive the application using a scripting language.
Note: If you are unfamiliar with Microsoft DOS, Applied Biosystems recommends
running the application from the user interface.
Running the
Software from a
Command Line
1. In the desktop, double-click My Computer > AppliedBiosystems > SDS2.2.1.
2. Create a copy of the SDS 2,bat file and rename it (such as SDS_RESULTS.bat).
3. Using the Notepad application, edit the contents of the batch file as follows.
Example Code A-17
SDS2.bat Batch File
The text of the batch file appears as follows:
set classpath=./service/config;./service/Lib/audit.jar;./service/Lib/rtc.jar;
./service/Lib/ab-reqres.jar;./service/Lib/translation.jar;./service/Lib/apv.jar;
./service/lib/coreutil.jar;./service/JMS/lib/fmprtl.zip;./service/Lib/coregui.jar;
./service/lib/EmlException.jar;./service/lib/emlhandler.jar;./service/lib/esig.jar;
./service/lib/esig/exception.jar;./service/lib/Coreutil.jar;
set path=./lib/algorithm/bin/win32;./lib;%path%
jre\bin\java -Dab.home=C:\AppliedBiosystems\SDS2.2\service -Dcom.apldbio.sds.global.approot=.
-Dcom.apldbio.sds.global.config= .\config\config.properties -Xms110m -Xmx300m
-Dsource=batchfile -Ddefport=8080 -classpath %classpath%;lib\SDS2.jar;lib\common.jar;
lib\sdsdb.jar;lib\ojdbc14.jar;lib\accesscontrol.jar;lib\sdsdb.jar;lib\activation.jar;
lib\mail.jar;lib\accesscontrol.jar;lib\Coreutil.jar;lib\EmlException.jar
com.apldbio.sds.shell.SDSMain
In the last line, replace
com.apldbio.sds.shell.SDSMain
with one of the following, to generate:
• Multicomponent data for an SDS plate document file, enter:
com.apldbio.sds.MulticompAnalyzerMain %*
• Analyzed data for an SDS plate document file, enter:
com.apldbio.sds.SDSResultsAnalyzerMain %*
• Multicomponent and analyzed data for a plate document file, enter:
com.apldbio.sds.MulticompAndResultsAnalyzerMain %*
4. Save and close the batch file.
5. Invoke the file at the command line by entering:
SDS_RESULTS.bat sdsfile.sds sdsresultsfile.txt
where:
• SDS_RESULTS.bat – Is the name of the batch file you created.
• sdsfile.sds – Is the SDS plate document file that you have targeted for
analysis.
• sdsresultsfile.txt – Is the name of the exported results file.
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Operating the SDS Software from a Command Line
Enhancing the
Performance of
the SDS Software
It is possible to enhance the performance (processing speed) of the SDS software by
increasing the memory partition allocated for the software. To increase the memory
made available to the SDS software, insert the parameters shown in the example
below into any command issued to the software.
jre\bin\java -Dab.home=C:\AppliedBiosystems\SDS2.2\service
-Dcom.apldbio.sds.global.approot=. -Dcom.apldbio.sds.global.config=
.\config\config.properties -Xms80m -Xmx90m -Dsource=batchfile
-Ddefport=8080…
where:
• -Xms80m – Is the minimum amount of memory that the operating system will
allocate for the SDS software.
• -Xmx90m – Is the maximum amount of memory that the operating system will
allocate for the SDS software.
Applied Biosystems recommends the following settings:
• End-point runs (allelic discrimination): -Xms20m -Xmx60m
• Real-time runs (absolute or relative quantifiation): -Xms80m -Xmx90m
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Appendix A Software Reference
Using the Search Tool
Programming
Filters
The Search tool helps to organize the large set data in the Add files dialog box. The
Search tool allows the software to display a subset of the total number of plate
documents based on a set of predefined criteria.
The Search tool operates on a logic similar to a simple computer programming
language. Each filter consists of a single logical statement that instructs the software
to include or exclude plate documents based on a specific parameter or quality. The
statements can be concatenated to screen the displayed data based on multiple
criteria.
Search
Statements
The Search tool consists of a table in which each row represents a single statement
that tests the associated data set for a condition. Every time a row (or statement) is
added to the table, the software evaluates each plate document in the selected
directory for the condition defined by the statement. If a plate document meets the
criteria defined by the test (the test evaluates true), then the software displays it in the
Search Results list. If a plate document does not meet the criteria (the test evaluates
false), the software hides the document from view and excludes the data from the
analysis. As explained later in this section, the Search tool can evaluate multiple
expressions (many rows) to refine the data set.
Search Statement
Syntax
Figure A-1 shows a filter created in the Add Files dialog box that illustrates the basic
structure of all statements.
A-20
Logic Operator
A logical operator
(IF, AND, or OR)
Column Category
Describes the category
for the evaluation
Figure A-1
Add Files dialog box
DRAFT
Conditional Operator
Describes the nature of
the evaluation
Value Criteria
Contains the condition
for the evaluation
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Using the Search Tool
Logical Operators
(IF, AND, OR)
Every statement begins with one of three logical operators: IF, OR, or AND. The
logical operator determines the relationship between the statements in the Add Files
dialog box.
Logical IF Statements
By default, the first statement in the table is always an IF statement. If a plate
document meets the criteria defined by the statement, it is displayed within the Add
Files dialog box.
Logical AND Statements
A logical AND statement evaluates the data set for two expressions: the expression
defined by the AND statement, and the expression defined by the statement in the
previous table row. When an AND statement is used in a filter table, the software
displays a plate document if it evaluates true for both expressions.
Logical OR Statements
Similar to the logical AND statement, a logical OR statement also evaluates the data set
for two expressions (both the previous and current table rows). When an OR statement
is used in a Filter table, the software displays a plate document if it evaluates true for
either expression (or both).
Multiple AND and OR Statements
If dealing with a very large data set, you may want to further refine the number of
plate documents retrieved in a search by creating multiple filter statements. When
creating multiple statements, remember to create and apply them one at a time to
ensure that they function as expected. Also, remember that the software applies each
filter in the order listed in the Add Files dialog box.
Column
Categories
Condition
Operators
Each statement evaluates plate documents based on the values of specific plate
document attributes.
The Condition operator establishes the relationship between the Column category
and the Value 1 and Value 2 criteria by defining the action of the evaluation. The
following list summarizes the possible conditional operators and their corresponding
actions.
•
•
•
•
•
•
•
•
Value 1 and 2
Criteria
> : Greater than Value 1
>= : Greater than or equal to Value 1
< : Less than Value 1
<= : Less than or equal to Value 1
equals : Equal to Value 1
< > : Does not equal Value 1
between : Is greater than Value 1 but less than Value 2
contains : Contains the alphanumeric string found in Value 1
The Value 1 and Value 2 criteria define the condition(s) for the evaluation.
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Appendix A Software Reference
Connecting SDS Software to the Database
Required
Information
IMPORTANT! Only users belonging to the Administrator or Service Engineer user
group can modify the database parameters for an SDS client application.
You need the following information to connect a SDS client application to the database:
• Host Name – The Host Name setting or the IP address for the SDS Enterprise
Database. Check with your database administrator for the correct identification
notation for the server running the database on your network.
• Port Number – The Port Number default setting is 1521. (You may need to
check with your database administrator if the port setting has been modified.)
• Service Name – The Service Name setting is configured during the installation
of the SDS Enterprise Database. The default is SDS22.
Disabling/Enabling
the Database
Functions in the
SDS Software
The SDS software allows users of the Administrator User Group to disable the
database functions of the software. When disabled, the software hides the toolbar
buttons and menu commands for opening and saving data to the database.
Note: The enable/disable feature can be used during periods when the network
connection is unavailable. By disabling the database connection, the SDS software
does not require users to log in before using the 7900HT instrument. After the
database becomes available again, the data collected in the interim can be uploaded
to the SDS Enterprise Database using the SDS Document Loader. For more
information, see the SDS Enterprise Database for the Applied Biosystems 7900HT
Fast Real-Time PCR System Administrators Guide (P/N 4351669).
To disable or enable the database features of the SDS software:
IMPORTANT! You must belong to the Administrators User Group to modify the
database options.
1. Start the SDS software (see page 2-7 for more information).
2. In the Login dialog box, enter a user name and password of a user account that
belongs to the Administrator user group (see the SDS Enterprise Database for
the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators
Guide for more information on user accounts).
3. Select Tools > Options.
4. In the Options dialog box, select the Database tab.
5. Select or deselect the Enable Enterprise Options check box to activate or
deactivate the database features.
6. Click
.
7. When prompted, enter your user name and password, then click
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Connecting SDS Software to the Database
Reconfiguring the
Database
Connection
IMPORTANT! You must have Administrative privileges to reconfigure the SDS
Enterprise Database connection (see the SDS Enterprise Database for the Applied
Biosystems 7900HT Fast Real-Time PCR System Administrators Guide for more
information on user accounts).
The connection to the SDS Enterprise Database can be reconfigured from the
Options dialog box of the SDS software as explained below.
1. Start the SDS software (see page 2-7 for more information).
2. In the Login dialog box, enter a user name and password of a user account that
belongs to the Administrator user group.
3. Select Tools > Options.
4. In the Options dialog box, select the Database tab.
5. Configure the Current Selected Connection settings:
Host Name field
Enter the host name or
IP address of the database
Port Number field
Enter the port number of the
database connection
(default is 1521)
Service Name field
Enter the name of the database
(default is SDS22)
6. Test the new connection:
a. Click
to test the new connection.
The software briefly checks that a database is at the given IP address. If the
connection is successful, the software displays the Login dialog box.
b. In the Test Login dialog box, enter your User Name and Password, then
click
.
• If the SDS software is unable to successfully connect to the database, the
software displays the error message shown below.
7. In the Options dialog box, click
.
8. Log into the database by entering your user name and password, then
click
.
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Appendix A Software Reference
A-24
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Designing TaqMan Reagent-Based
Assays
B
In This Appendix
B
Assay Development Guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2
Design Tips for Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5
Design Tips for Quantitative PCR Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-6
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B-1
Appendix B Designing TaqMan Reagent-Based Assays
Assay Development Guidelines
TaqMan ReagentBased Assay
Development
Program
1. Identify target sequence(s) (see page B-2).
2. Design the TaqMan® probes and the forward and reverse primers (see page B-2).
3. Order reagents.
4. Quantitate the concentrations of the probes and primers (see page B-3).
5. Prepare the master mix (see page B-3).
6. Optimize the primer concentrations (see page B-3).
7. Run the assay (see page B-4).
Identify Target
Sequence(s)
A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotide
sequence of interest. For optimal results, the target template should meet the
following requirements:
• The target nucleotide sequence must contain binding sites for both primers
(forward and reverse) and the fluorogenic probe.
• Short amplicons work best. Amplicons ranging from 50–150 bp typically yield
the most consistent results.
• If designing assays for quantitative PCR, see “Design Tips for Quantitative PCR
Assays” on page B-6 for additional recommendations.
Design Probes
and Primers
The following sections contain general guidelines for designing primers and probes.
For specific design tips, refer to the appropriate section: for Allelic Discrimination
see page B-5 and for Quantitative PCR see page B-6.
Design Probe(s) for the Assay
Adhere to the following guidelines when designing TaqMan probes:
• Keep the G-C content in the range of 30–80%.
• Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more Gs should be avoided).
• No G on 5´ end.
• Keep the melting temperature (Tm) in the range of 68-70 °C for quantitative PCR
and 65-67 °C for allelic discrimination (using the Primer Express™ software).
• Select the strand that gives the probe with more Cs than Gs.
• For allelic discrimination (see page B-5):
– Adjust probe length so that both probes have the same Tm.
– Position the polymorphism site approximately in the center of each probe.
• For absolute or relative quantification (see page B-6)
• For multiplex PCR applications (involving multiple probes), design the probes
with different fluorescent reporter dyes (see Table B-1).
B-2
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Assay Development Guidelines
Table B-1
Reporter Dyes for Multiplex PCR Applications
Reporter Dye*
First Probe
Second Probe
FAM™
VIC®
*The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest
degree of spectral separation.
Design Primers for the Assay
Adhere to the following guidelines when designing primers for 5´-nuclease assays:
• Keep the G-C content in the range of 30–80%.
• Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more bases should be avoided).
• Keep the Tm in the range of 58-60 °C (using the Primer Express™ software).
• Limit the G and/or C bases on the 3´ end. The five nucleotides at the 3´ end
should have no more than two G and/or C bases.
• Place the forward and reverse primers as close as possible to the probe without
overlapping the it.
• Use an annealing temperature of 58-60 °C for quantitative PCR, and 58-60 °C for
allelic discrimination (except for TaqMan® PDARs for Allelic Discrimination).
Quantitate the
Probes and
Primers
Use a spectrophotometric method to determine the concentrations of the probes and
primers received. See the TaqMan® Universal PCR Master Mix Protocol
(PN 4304449) or the TaqMan® Fast Universal PCR Master Mix (2✕) Protocol
(PN 4351891) for specific information about primer and probe quantification.
Prepare
Master Mix
Refer to the TaqMan® Universal PCR Master Mix Protocol (PN 4304449) or the
TaqMan® Fast Universal PCR Master Mix (2✕) Protocol (PN 4351891) for specific
information about preparing the master mix for use.
IMPORTANT! PCR master mix used with the 7900HT instrument must contain a
passive reference dye. The SDS software uses the signal from the passive reference
to normalize the reporter fluorescence making well-to-well comparisons possible.
All Applied Biosystems master mix products contain an optimal concentration of the
ROX passive reference dye.
Note: Applied Biosystems protocols are available on the Applied Biosystems
Company Web Site. See “How to Obtain Services and Support” on page xii for more
information.
Optimize
Primer/Probe
Concentrations
Refer to the TaqMan® Universal PCR Master Mix Protocol (PN 4304449) for
specific information about optimizing primer/probe concentrations.
Note: Applied Biosystems protocols are available on the Applied Biosystems
Company Web Site. See “How to Obtain Services and Support” on page xii for more
information.
Run Your Custom
Assay
Note: If conducting a quantitative PCR experiment, consider the use of replicate
assays to enhance the precision of you data.
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B-3
Appendix B Designing TaqMan Reagent-Based Assays
Design Tips for Allelic Discrimination Assays
Discrimination by
Multiple Probes
TaqMan Probe
Design
Guidelines
By using different reporter dyes, cleavage of multiple probes can be detected in a
single PCR. One application of this multi-probe capability is to use allele-specific
probes to distinguish genetic polymorphisms (Bloch, 1991, Lee et al., 1993). Probes
that differ by as little as a single nucleotide will exhibit allele-specific cleavage. This
is true even for probes with a reporter on the 5' end and the non-fluorescent quencher
on the 3´ end (Bloch, 1991).
IMPORTANT! When designing probes, it is important to consider probes from both
strands.
Follow the guidelines in the table below for designing TaqMan MGB probes:
Table B-2 Guidelines for Designing TaqMan MGB Probes for Allelic Discrimination
Priority
Guideline
Avoid probes with a guanine residue at the 5´ end of the probe.
1
A guanine residue adjacent to the reporter dye will quench the reporter
fluorescence, even after cleavage.
2
Select probes with a Primer Express software–estimated Tm of 65–67 °C.
3
Make the TaqMan MGB probes as short as possible, but no fewer than
13 nucleotides in length.
Avoid runs of an identical nucleotide.
4
This is especially true for guanine, where runs of four or more should be
avoided.
Position the polymorphic site in the central third of the probe.
Note: The polymorphic site can be shifted toward the 3´ end to meet the
above guidelines, however, the site must be located more than two
nucleotides upstream from the 3´ terminus.
The following figure illustrates the placement of a polymorphism in an example
probe (N = Nucleotide).
First, try to position the polymorphic
site in the central third of the probe.
5
5´
3´
N
N N N N N N N N N N N N N N N N N N N N
Polymorphism
B-4
Do not place
it here.
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If necessary, place the
polymorphism here.
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Design Tips for Quantitative PCR Assays
Design Tips for Quantitative PCR Assays
Selecting an
Amplicon Site for
Gene Expression
Assays
Selecting a good amplicon site ensures amplification of the target mRNA without
co-amplifying the genomic sequence, pseudogenes, and related genes.
Applied Biosystems recommends the following guidelines when selecting an
amplicon site for quantification assays:
• Primers and probes must be designed following the “Assay Development
Guidelines” on page B-2.
• The amplicon should span one or more introns to avoid amplification of the
target gene in genomic DNA.
• The primer pair has to be specific to the target gene and does not amplify
pseudogenes or other related genes.
• Test amplicons and select those that have the highest signal-to-noise ratio (such
as those yielding low CTs with cDNA and no amplification with no template
control or genomic DNA).
• If no good sequence is found, it may be necessary to examine the sequence and
redesign the amplicon or simply screen for more sites.
If the gene you are studying does not have introns, then you cannot design an
amplicon that will amplify the mRNA sequence without amplifying the genomic
sequence. In this case, it may be necessary to run RT minus controls.
Selecting and
Preparing
Standards for
Absolute
Quantification
To ensure accurate results, the standards used for absolute quantification must be
carefully engineered, validated, and quantified before use. Consider the following
critical points for the proper use of absolute standard curves:
• The DNA or RNA used must be a single, pure species. For example, plasmid
DNA prepared from E. coli often is contaminated with RNA, which increases
the A260 measurement and inflates the copy number determined for the plasmid.
• In general, DNA cannot be used as a standard for absolute quantification of
RNA because there is no control for the efficiency of the reverse transcription
step.
• Absolute quantities of the standard must be known by some independent means.
Plasmid DNA or in vitro transcribed RNA are commonly used to prepare
absolute standards. Concentration is measured by A260 and converted to the
number of copies using the molecular weight of the DNA or RNA.
• Consider the stability of the diluted standards, especially for RNA. Divide
diluted standards into small aliquots, store at -80 °C, and thaw only once before
use. An example of the effort required to generate trustworthy standards is
provided by Collins et al. (Anal. Biochem. 226:120-129, 1995), who reported
on the steps they used in developing an absolute RNA standard for viral
quantification.
• Pipetting must be accurate because the standards must be diluted over several
orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be
concentrated in order to measure an accurate A260 value. The concentrated DNA
or RNA must then be diluted 106 –1012 -fold to be at a concentration similar to
the target in biological samples.
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B-5
Appendix B Designing TaqMan Reagent-Based Assays
B-6
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Kits, Reagents, and Consumables
In This Appendix
C
C
Interchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . .C-2
Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-3
Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-5
TaqMan Pre-Developed Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-6
Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-6
Note: Part numbers listed in this appendix are for customers inside the United
States. Contact your Regional Sales Office for local Part numbers and prices. See
“How to Obtain Services and Support” on page xii.
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C-1
Appendix C Kits, Reagents, and Consumables
Interchangeable Sample Block Modules and Accessories
The 7900HT instrument features a Peltier-based, interchangeable sample block module
based on the technology established in the GeneAmp® PCR System 9700 thermal cycler.
The use of an interchangeable sample block module:
• Reduces instrument downtime by allowing immediate replacement of the block.
• Permits easy access to the sample block for troubleshooting and maintenance
(see page 7-14).
• Supports multiple consumable formats.
• Provides several different modes of operation (including Max mode and
programmable temperature ramps).
Table C-1
Sample Block Modules and Accessories
Part No.
Description
4331406
7900HT System Standard 384-Well Block Upgrade Kit
Quantity
1 kit
Includes a Standard 384-Well Block, a 384-well plate adapter,
and a Sequence Detection Systems 384-Well Spectral
Calibration Kit (PN 4323977)
4331405
7900HT System Standard 96-Well Block Upgrade Kit
1 kit
Includes a Standard 96-Well Block, a 96-well plate adapter,
and an ABI PRISM™ 7900HT Sequence Detection Systems
96-Well Spectral Calibration Kit (PN 4328639)
4351402
7900HT System Fast 96-Well Block Upgrade Kit
1 kit
Includes a Fast 96-Well Block, a Fast 96-Well Plate Adapter, a
7900HT System Fast 96-Well Spectral Calibration Kit
(PN 4351653), a TaqMan® RNase P Fast 96-Well Instrument
Verification Plate (PN 4351979), and a TaqMan® Fast
Reagents Starter Kit (PN 4352407)
4329012
C-2
7900HT System TaqMan® Low Density Array Upgrade
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Consumables and Disposables
Consumables and Disposables
The Applied Biosystems 7900HT Fast Real-Time PCR System can run:
• ABI PRISM™ 384-Well Optical Reaction Plates sealed with optical adhesive
covers
• ABI PRISM™ 96-Well Optical Reaction Plates sealed with optical adhesive
covers or ABI PRISM™ Optical Caps (flat cap strips only)
• Optical 96-Well Fast Thermal Cycling Plates sealed with optical adhesive
covers
• TaqMan® Low Density Arrays
The optical plates recommended above are designed specifically for
fluorescence-based PCR chemistries and are frosted to minimize external fluorescent
contamination. Before running prepared optical plates on the 7900HT instrument,
each plate must be sealed with the optical adhesive covers recommended above.
Applied Biosystems optical adhesive covers are specifically designed to permit the
transmission of light to and from the wells of the optical plate.
IMPORTANT! Do not use MicroAmp® Optical Caps or MicroAmp® Optical Tubes
with the 7900HT instrument. The instrument is not designed to run MicroAmp
consumables which may damage its internal components if used.
Table C-2
Part No.
Consumables and Disposables for the 7900HT Instrument
Description
Quantity
Optical Adhesive Covers
4313663
ABI PRISM™ Optical Adhesive Cover Starter Kit
20 Covers
Includes 20 ABI PRISM Optical Adhesive Covers, an
Applicator, and an ABI PRISM™ Optical Cover Compression
Pad.
™
4311971
ABI PRISM™ Optical Adhesive Covers
100 Covers
4360954
Optical Adhesive Covers
25 Covers
4323032
ABI PRISM™ Optical Caps, 8 Caps/Strip
300 Strips/
Pkg
2400 Caps/
Pkg
384-Well Optical Reaction Plates
4309849
ABI PRISM™ 384-Well Clear Optical Reaction Plate with
Barcode (code 128)
50 Plates
4326270
ABI PRISM™ 384-Well Clear Optical Reaction Plate with
Barcode (code 128), 10-Pack
500 Plates
Includes 10 of PN 4309849, ABI PRISM™ 384-Well Clear
Optical Reaction Plates with Barcode
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C-3
Appendix C Kits, Reagents, and Consumables
Table C-2
Part No.
Consumables and Disposables for the 7900HT Instrument
Description
Quantity
96-Well Optical Reaction Plates
4306737
ABI PRISM™ 96-Well Optical Reaction Plate with Barcode
(code 128)
20 Plates
4326659
ABI PRISM™ 96-Well Optical Reaction Plate with Barcode
(code 128), 25-Pack
500 Plates
Includes 25 of PN 4306737, ABI PRISM™ 96-Well Optical
Reaction Plates with Barcode
4314320
ABI PRISM™ 96-Well Optical Reaction Plate with Barcode
(code 128) and ABI PRISM™ Optical Adhesive Covers
100 Plates
100 Covers
Includes 100 ABI PRISM™ Optical Adhesive Covers
(PN 4311971) and 5 of PN 4306737, ABI PRISM™ 96-Well
Optical Reaction Plates with Barcode
4312063
MicroAmp® Splash Free Support Base for 96-Well Reaction
Plates
10 Bases
Optical 96-Well Fast Plates
C-4
4346906
Optical 96-Well Fast Thermal Cycling Plate with Barcode
(code 128)
20 Plates
4312063
MicroAmp® Splash Free Support Base for 96-Well Reaction
Plates
10 Bases
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Instrument Maintenance and Verification
Instrument Maintenance and Verification
The following sequence detection kits and reagents are used to perform routine
maintenance on and verify the function of the Applied Biosystems 7900HT Fast
Real-Time PCR System. For more information about the use of the kits below, see
Chapter 7, “Maintaining the Instrument.”
Table C-3
Part
Number
Consumables for Instrument Maintenance and Verification
Description
Quantity
Sequence Detection Systems Spectral Calibration Kits
4328639
ABI PRISM™ 7900HT Sequence Detection Systems 96-Well
Spectral Calibration Kit
3 x 96-Well
Plates
Includes three ABI PRISM™ 96-Well Optical Reaction Plates:
one preloaded and sealed background plate, and two
preloaded and sealed Spectral Calibration plates containing
eight separate dye standards (FAM™, JOE™, NED™, ROX™,
SYBR® Green, TAMRA™, TET™, VIC®).
4323977
Sequence Detection Systems 384-Well Spectral Calibration
Kit
Includes two ABI PRISM™ 384-Well Optical Reaction Plates:
one preloaded and sealed background plate and one
preloaded and sealed Spectral Calibration plate containing
eight separate dye standards (FAM, JOE, NED, ROX, SYBR
Green, TAMRA, TET, VIC).
4351653
7900HT System Fast 96-Well Spectral Calibration Kit
Includes three Optical 96-Well Fast Thermal Cycling Plates:
one preloaded and sealed background plate, and two
preloaded and sealed pure dye plates containing eight
separate dye standards (FAM™, JOE™, NED™, ROX™, SYBR®
Green, TAMRA™, TET™, VIC®).
2x
384-Well
Plates
3 x 96-Well
Plates
TaqMan RNase P Instrument Verification Plates
4310982
TaqMan® RNase P Instrument Verification Plate
Includes one ABI PRISM 96-Well Optical Reaction Plate. Each
well contains preloaded reaction mix (master mix, RNase P
primers, and FAM™ dye-labeled probe) and template to detect
and quantitate genomic copies of the human RNase P gene.
™
4323306
TaqMan® RNase P 384-Well Instrument Verification Plate
Includes one ABI PRISM 384-Well Optical Reaction Plate.
Each well contains preloaded reaction mix (master mix,
RNase P primers, and FAM™ dye-labeled probe) and template
to detect and quantitate genomic copies of the human
RNase P gene.
™
1 x 96-Well
Plate
1x
384-Well
Plate
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C-5
Appendix C Kits, Reagents, and Consumables
Table C-3
Consumables for Instrument Maintenance and Verification
Part
Number
Description
Quantity
4351979
TaqMan® RNase P Fast 96-Well Instrument Verification Plate
1 x 96-Well
Plate
Includes one Optical 96-Well Fast Thermal Cycling Plate.
Each well contains preloaded reaction mix (master mix,
RNase P primers, and FAM™ dye-labeled probe) and template
to detect and quantitate genomic copies of the human
RNase P gene.
TaqMan® Low Density 7900HT Installation Array (TGF-β card)
TaqMan Pre-Developed Assays and Reagents
For the latest information on TaqMan PDARs covering gene expression
quantification and allelic discrimination, visit the TaqMan PDAR list on the
Applied Biosystems web site at:
• www.appliedbiosystems.com/pdarlist
Custom Oligonucleotide Synthesis
To order custom oligonucleotides:
• Visit the Applied Biosystems Online Store
(http://store.appliedbiosystems.com), or
• Email Applied Biosystems with your order
([email protected])
Table C-4
Custom Oligonucleotides
Part
Number
Description
TaqMan® MGB Probes (5’-Fluorescent label: 6-FAM, VIC or TET)
4316034
5,000-6,000 pmols
4316033
15,000-25,000 pmols
4316032
50,000-100,000 pmols
TaqMan® Probes (5’-Fluorescent label: 6-FAM, VIC or TET; 3’-label: TAMRA)
C-6
450025
5,000-6,000 pmols
450024
15,000-25,000 pmols
450023
50,000-100,000 pmols
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Custom Oligonucleotide Synthesis
Table C-4
Custom Oligonucleotides
Part
Number
Description
Sequence Detection Primers
4304970
Minimum 4,000 pmols purified for sequence detection
4304971
Minimum 40,000 pmols purified for sequence detection
4304972
Minimum 130,000 pmols purified for sequence detection
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C-7
Appendix C Kits, Reagents, and Consumables
C-8
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Theory of Operation
In This Appendix
D
D
Fluorescent-Based Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Real-Time Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-4
Comparative CT Method of Relative Quantification. . . . . . . . . . . . . . . . . . . . . . . D-8
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D-1
Appendix D Theory of Operation
Fluorescent-Based Chemistries
Fundamentals of
the 5´ Nuclease
Assay
The PCR reaction exploits the 5´ nuclease activity of AmpliTaq Gold® DNA
Polymerase to cleave a TaqMan® probe during PCR. The TaqMan probe contains a
reporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe.
During the reaction, cleavage of the probe separates the reporter dye and the
quencher dye, which results in increased fluorescence of the reporter. Accumulation
of PCR products is detected directly by monitoring the increase in fluorescence of
the reporter dye. Figure D-1 shows the forklike-structure-dependent,
polymerization-associated 5´–3´ nuclease activity of AmpliTaq Gold DNA
Polymerase during PCR.
Polymerization
5'
3'
5'
R
Foward
Primer
Probe
Q
3'
5'
Strand displacement
Reverse
Primer
R
Probe
5'
3'
5'
Cleavage
R = Reporter
Q = Quencher
Q
3'
5'
3'
5'
R
Probe
5'
3'
5'
Q
3'
5'
3'
5'
Polymerization completed
R
5'
3'
5'
Figure D-1
3'
5'
Probe
Q
3'
5'
3'
5'
5´–3´ Nuclease Activity of AmpliTaq Gold DNA Polymerase
When the probe is intact, the proximity of the reporter dye to the quencher dye results
in suppression of the reporter fluorescence primarily by Förster-type energy transfer
(Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, the
probe specifically anneals between the forward and reverse primer sites.
The 5´–3´ nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the
probe between the reporter and the quencher only if the probe hybridizes to the
target. The probe fragments are then displaced from the target, and polymerization of
the strand continues. The 3´ end of the probe is blocked to prevent extension of the
probe during PCR. This process occurs in every cycle and does not interfere with the
exponential accumulation of product.
The increase in fluorescence signal is detected only if the target sequence is
complementary to the probe and is amplified during PCR. Because of these
requirements, any nonspecific amplification is not detected.
D-2
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Fluorescent-Based Chemistries
Basics of SYBR
Green Dye
Chemistry
The SYBR® Green 1 Double-Stranded Binding Dye is used for the fluorescent
detection of double-stranded DNA (dsDNA) generated during PCR. The SYBR
Green 1 Dye binds non-specifically to dsDNA and generates an excitation-emission
profile similar to that of the FAM™ reporter dye. When used in combination with a
passive reference, the SYBR Green 1 Dye can be employed to perform several
SDS-related experiments including quantitative PCR and dissociation cure analysis.
Figure D-2 illustrates the action of the SYBR Green 1 dye during a single cycle of a
PCR.
5´
3´
3´
5´
When added to the reaction, the SYBR Green 1 Dye binds non-specifically to
the hybridized dsDNA and fluoresces
Dissociation
3´
5´
5´
3´
Denaturation complete, the SYBR Green 1 Dye dissociates from the strand,
resulting in decreased fluorescence
Polymerization
5´
3´
Forward
Primer
5´
5´
Reverse
Primer
3´
5´
During the extension phase, the SYBR Green 1 Dye begins binding to the
PCR product
Polymerization Complete
3´
5´
5´
3´
3´
5´
5´
3´
Polymerization is complete and SYBR Green 1 Dye is completely bound,
resulting in a net increase in fluorescence
Figure D-2
Binding Activity of the SYBR Green 1 Dye during the PCR
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D-3
Appendix D Theory of Operation
Real-Time Data Analysis
Kinetic Analysis/
Quantitative PCR
The 7900HT instrument can be used to determine the absolute or relative quantity of
a target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle change
in fluorescence signal as a result of amplification during a PCR. This form of
quantitative PCR analysis, called “kinetic analysis,” was first described using a
non-sequence-specific fluorescent dye, ethidium bromide, to detect PCR product
(Higuchi et al., 1992; Higuchi et al., 1993). The use of TaqMan probes and reagents
further enhances the method by providing sequence-specific amplification of
multiple targets for ‘comparative’ or ‘relative’ quantification. The fewer cycles it
takes to reach a detectable level of fluorescence, the greater the initial copy number
of the target nucleic acid.
Amplification Plot
8.0
Phase 3, plateau
2.0
1.0
Phase 2, linear
Rn
Phase 1, geometric
0.0
0
10
20
30
40
Cycle Number
Figure D-3
Phases of a Typical Amplification Curve
When graphed in real-time on a linear scale, normal amplification of PCR product
generates a curve similar to the one shown in Figure D-3. This ‘amplification’ curve
consists of three distinct regions that characterize the progression of the PCR.
Phase 1: Geometric (Exponential)
Detection of the high-precision geometric phase is the key to high-precision
quantitative PCR. The geometric phase is a cycle range of high precision during
which amplification is characterized by a high and constant efficiency. It occurs
between the first detectable rise in fluorescence and before the beginning of the
linear phase. When plotted on a log scale of DNA vs. cycle number, the curve
generated by the geometric phase should approximate a straight line with a slope.
The 7900HT instrument typically delivers sufficient sensitivity to detect at least
3 cycles in the geometric phase, assuming reasonably optimized PCR conditions.
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Real-Time Data Analysis
Phase 2: Linear
The linear phase is characterized by a leveling effect where the slope of the
amplification curve decreases steadily. At this point, one or more components have
fallen below a critical concentration and the amplification efficiency has begun to
decrease. This phase is termed linear, because amplification approximates an
arithmetic progression, rather than a geometric increase. Because the amplification
efficiency is continually decreasing during the Linear phase, it exhibits low
precision.
Phase 3: Plateau
Finally, the amplification curve achieves the plateau phase at which time the PCR
stops and the Rn signal remains relatively constant.
Determining
Initial Template
Concentration
and Cycle
Number
At any given cycle inside the geometric phase of PCR, the amount of product is
proportional to the initial number of template copies. When one template is diluted
several times, as with the RNase P target in the RNase P Instrument Verification
Plates (see Appendix C), the ratio of template concentration to detectable signal is
preserved in the exponential phase for all dilutions (see Figure D-4). This
relationship appears to change as rate of amplification approaches a plateau.
8.0
2.0
1.0
Rn
20000
10000
5000
2500
1250
0.0
0
10
20
30
40
Cycle Number
Figure D-4 Amplification Plot from a Real-Time Run of an RNase P Instrument
Verification Plate
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D-5
Appendix D Theory of Operation
Fluorescence vs.
Amplified
Product
When using TaqMan fluorogenic probes with the 7900HT instrument, fluorescence
emission increases in direct proportion to the amount of specific amplified product.
As Figure D-4 on page D-5 demonstrates, the graph of normalized reporter (Rn) vs.
cycle number during PCR appears to have three stages. Initially, Rn appears as a flat
line because the fluorescent signal is below the detection limit of the 7900HT
instrument. In the second stage, the signal can be detected as it continues to increase
in direct proportion to the increase in the products of PCR. As PCR product
continues to increase, the ratio of AmpliTaq Gold polymerase to PCR product
decreases. When template concentration reaches 10-8 M, PCR product ceases to grow
exponentially. This signals the third stage of Rn change, which is roughly linear and
finally reaches a plateau at about 10-7 M (Martens and Naes, 1989).
The progressive cleavage of TaqMan fluorescent probes during the PCR makes
possible the correlation between initial template concentration and the rise in
fluorescence. As the concentration of amplified product increases in a sample, so
does the Rn value. During the exponential growth stage (the geometric phase), the
relationship of amplified PCR product to initial template can be shown in the
following equation:
Nc = N ( 1 + E ) c
where Nc is the concentration of amplified product at any cycle, N is the initial
concentration of target template, E is the efficiency of the system, and c is the cycle
number.
Calculating
Threshold Cycles
The Applied Biosystems 7900HT Fast Real-Time PCR System creates quantifiable
relationships between test samples based on the number of cycles elapsed before
achieving detectable levels of fluorescence. Test samples containing a greater initial
template number cross the detection threshold at a lower cycle than samples
containing lower initial template. The SDS software uses a Threshold setting to
define the level of detectable fluorescence.
The threshold cycle (CT) for a given amplification curve occurs at the point that the
fluorescent signal grows beyond the value of the threshold setting. The CT represents
a detection threshold for the 7900HT instrument and is dependent on two factors:
• Starting template copy number
• Efficiency of DNA amplification the PCR system
How the SDS Software Determines CTs
To determine the CT for an Amplification plot, the SDS software uses data collected
data from a predefined range of PCR cycles called the ‘baseline’ (the default
baseline occurs between cycles 3 and 15). First, the software calculates a
mathematical trend based on the baseline cycles’ Rn values to generate a baseline
subtracted Amplification plot of ∆Rn versus cycle number. Next, an algorithm
searches for the point on the Amplification plot at which the ∆Rn value crosses the
threshold setting (the default threshold setting is 0.2). The fractional cycle at which
the intersection occurs is defined as the threshold cycle (CT) for the plot.
Note: It may be necessary to adjust the baseline and threshold settings to obtain
accurate and precise data. For further information on resetting the baseline and
threshold settings, see “Setting the Baseline and Threshold Values” on page 6-46.
D-6
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Real-Time Data Analysis
Significance of
Threshold Cycles
Beginning with the equation describing the exponential amplification of the PCR:
Xn = Xm ( 1 + EX )
n–m
where:
Xn
=
number of target molecules at cycle n (so that n ≥ m)
Xm
=
number of target molecules at cycle m (so that m ″ n)
EX
=
efficiency of target amplification (between 0–1)
n-m
=
number of cycles elapsed between cycle m and cycle n
Amplicons designed and optimized according to Applied Biosystems guidelines
(amplicon size <150 bp) have amplification efficiencies that approach 100 percent.
Therefore EX=1 so that:
n–m
Xn = Xm ( 1 + 1 )
n–m
= Xm ( 2 )
To define the significance in amplified product of one thermal cycle, set n - m = 1 so
that:
X1 = X0 ( 2 )
= 2X 0
1
Therefore, each cycle in the PCR reaction corresponds to a two-fold increase in
product. Likewise, a change in threshold cycle number of one must equate to a
two-fold difference in initial template concentration.
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D-7
Appendix D Theory of Operation
Comparative CT Method of Relative Quantification
Arithmetic
Formula
The Comparative CT Method is similar to the Standard Curve Method. However, it
uses arithmetic formulas to achieve the same results for relative quantification. The
amount of target, normalized to an endogenous reference and relative to a calibrator,
is given by:
2
Derivation of the
Formula
– ∆∆C T
The equation that describes the exponential amplification of PCR is:
Xn = Xo × ( 1 + EX ) n
where:
Xn
= number of target molecules at cycle n
Xo
= initial number of target molecules
EX
= efficiency of target amplification
n
= number of cycles
The threshold cycle (CT) indicates the fractional cycle number at which the amount
of amplified target reaches a fixed threshold.
Thus,
XT = Xo × ( 1 + EX )
C T, X
= KX
where:
XT
CT,X
KX
D-8
= threshold number of target molecules
= threshold cycle for target amplification
= constant
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Comparative CT Method of Relative Quantification
A similar equation for the endogenous reference reaction is:
RT = Ro × ( 1 + ER )
C T, R
= KR
where:
RT
= threshold number of reference molecules
Ro
= initial number of reference molecules
ER
= efficiency of reference amplification
CT, R
KR
= threshold cycle for reference amplification
= constant
Dividing XT by RT gives the following expression:
C T, X
Xo × ( 1 + EX )
KX
X
------T- = --------------------------------------------- = -------- = K
C
KR
RT
T, R
Ro × ( 1 + ER )
The exact values of XT and RT depend on a number of factors, including:
•
•
•
•
•
Reporter dye used in the probe
Sequence context effects on the fluorescence properties of the probe
Efficiency of probe cleavage
Purity of the probe
Setting of the fluorescence threshold
Therefore, the constant K does not have to be equal to one.
Assuming efficiencies of the target and the reference are equivalent:
EX = ER = E,
C T, X – C T, R
X
------o × ( 1 + E )
=K
Ro
or
XN × ( 1 + E )
∆C T
=K
where:
XN
∆CT
= Xo/Ro, the normalized amount of target
= CT, X - CT, R, the difference in threshold cycles for target and reference
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D-9
Appendix D Theory of Operation
Rearranging gives the following expression:
XN = K × ( 1 + E )
– ∆C T
The final step is to divide the XN for any sample q by the XN for the calibrator (cb):
X
N, q
-------------X N, cb
– ∆C
T, q
– ∆∆C T
K × (1 + E )
- = (1 + E )
= --------------------------------------------– ∆C
K × (1 + E )
T, cb
where:
∆∆CT
= ∆CT,q – ∆CT,cb
For amplicons designed and optimized according to Applied Biosystems guidelines
(amplicon size <150 bp), the efficiency is close to 1. Therefore, the amount of target,
normalized to an endogenous reference and relative to a calibrator, is given by:
2
D-10
– ∆∆C T
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Limited Warranty Statement
Warranty
Statement
E
E
Applera Corporation, through its Applied Biosystems Group
(“Applied Biosystems“) warrants to the customer that, for a period ending on the
earlier of one year from the completion of installation or fifteen (15) months from the
date of shipment to the customer (the “Warranty Period”), the Applied Biosystems
7900HT Fast Real-Time PCR System purchased by the customer (the “Instrument”)
will be free from defects in material and workmanship, and will perform in
accordance with the installation specifications set forth in the system specifications
sheet which accompanies the instrument or which is otherwise available from an
Applied Biosystems sales representative.
During the Warranty Period, if the Instrument's hardware becomes damaged or
contaminated or if the Instrument otherwise fails to meet the Specifications,
Applied Biosystems will repair or replace the Instrument so that it meets the
Specifications, at Applied Biosystems' expense. However, if the thermal cycling
module becomes damaged or contaminated, or if the chemical performance of the
Instrument otherwise deteriorates due to solvents and/or reagents other than those
supplied or expressly recommended by Applied Biosystems, Applied Biosystems
will return the Instrument to Specification at the customer's request and at the
customer's expense. After this service is performed, coverage of the parts repaired or
replaced will be restored thereafter for the remainder of the original Warranty Period.
This Warranty does not extend to any Instrument or part which has been (a) the
subject of an accident, misuse, or neglect, (b) modified or repaired by a party other
than Applied Biosystems, or (c) used in a manner not in accordance with the
instructions contained in the Instrument User's Manual. This Warranty does not
cover the customer-installable accessories or customer-installable consumable parts
for the Instrument that are listed in the Instrument User's Manual. Those items are
covered by their own warranties.
Applied Biosystems' obligation under this Warranty is limited to repairs or
replacements that Applied Biosystems deems necessary to correct those failures of
the Instrument to meet the Specifications of which Applied Biosystems is notified
prior to expiration of the Warranty Period. All repairs and replacements under this
Warranty will be performed by Applied Biosystems on site at the Customer's
location at Applied Biosystems's sole expense.
No agent, employee, or representative of Applied Biosystems has any authority to
bind Applied Biosystems to any affirmation, representation, or warranty concerning
the Instrument that is not contained in Applied Biosystems's printed product
literature or this Warranty Statement. Any such affirmation, representation or
warranty made by any agent, employee, or representative of Applied Biosystems will
not be binding on Applied Biosystems.
Applied Biosystems shall not be liable for any incidental, special, or consequential
loss, damage or expense directly or indirectly arising from the purchase or use of the
Instrument. Applied Biosystems makes no warranty whatsoever with regard to
products or parts furnished by third parties.
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E-1
Appendix E Limited Warranty Statement
This Warranty is limited to the original location of installation and is not transferable.
THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE
INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED
WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED
WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR
PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF
APPLIED BIOSYSTEMS.
E-2
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Glossary
.sdd
The file extension of an ABI PRISM SDS Detector file for SDS software for the
7900HT instrument.
.sdm
The file extension of a SDS 7900HT Multiple Plate Document (*.sdm) for SDS
software for the 7900HT instrument.
.sds
The file extension of a SDS 7900HT Document (*.sds) for SDS software for the
7900HT instrument.
.sdt
The file extension of a SDS 7900HT Template Document (*.sdt) for SDS software for
the 7900HT instrument.
∆CT
The difference between the threshold cycle of a sample assay and the threshold cycle
of the corresponding endogenous reference: ∆CT = CT(target) - CT - CT (endogenous
control).
∆∆CT
For a given cDNA target, the difference between the average ∆CT value of a target
sample and the average ∆CT for the corresponding calibrator sample:
∆∆CT (test sample) = Avg∆CT (test sample) - Avg∆CT (calibrator sample). This value
is used to calculate expression fold value by the equation: Expression fold value =
2−∆∆C
T
∆Rn
The normalized reporter signal minus the baseline signal. The baseline is established
in the first few cycles of PCR. Like Rn, ∆Rn is not a fixed value but increases during
PCR when the amplicon copy number increases until the reaction approaches a
plateau.
5′ nuclease assay
The 5′ (five prime) nuclease assay in combination with the TaqMan® probes serves as
the basis for real-time polymerase chain reaction (PCR) technology on the 7900HT
instrument. See “Fundamentals of the 5´ Nuclease Assay” on page D-2 for a detailed
description of the reaction.
ABI PRISM SDS
Detector file (*.sdd)
The SDS detector file is a tab-delimited flat file that is used to transfer detector
information to and from the SDS software using the Export and Import functions of
the Detector Manager software. The file contains: (a) a header
“SDS2.2.1[GDF[Global Detector File”, (b) tab-delimited data for each detector (allele
Name, the name of the reporter dye, the name of the quencher dye, a description, a
comment, the owner, the creation date and time, the modification date and time, and
the color used to code the detector in graphical displays of the SDS software).
SDS 7900HT
Multiple Plate
Document (*.sdm)
Multiple plate documents are used for the analysis of relative quantification data.
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SDS 7900HT
Document (*.sds)
A file used by the SDS software to store plate documentation locally on a computer
hard drive. A plate document is a virtual representation of a consumable (plate) used
to contain samples and reagents during a sequence detection run. During the run, the
software uses the plate document to record the operation of the instrument (thermal
cycling and data collection parameters), organize and store the data gathered during
the PCR process, store the of the run data, and record analysis and display options.
SDS 7900HT
Template
Document (*.sdt)
Plate document templates have the same format as the SDS 7900HT Documents
(*.sds) but do not store raw data or bar codes.
absolute
quantification (AQ)
The objective of an absolute quantification experiment is to accurately determine the
absolute quantity of a single nucleic acid target sequence within an unknown sample.
The results of an absolute quantification experiment are reported in the same units of
measure as the standard used to make them.
allele
One of the variant forms of a gene at a particular locus or location on a chromosome
allelic
discrimination (AD)
The process by which two variants of a single nucleic acid sequence present in a
population of individuals' DNA samples is determined. Single nucleotide
polymorphisms (SNPs) comprise the most common variants that are assayed using
TaqMan reagents.
amplicon
A PCR amplified section of DNA
Amplification plot
The Amplification plot displays data from real-time PCR for gene expression assays
after signal normalization and multi-component analysis. The Amplification plot
appears only in plate documents containing analyzed data from real-time runs. It is
a 2D plot of normalized reporter fluorescence (for ∆Rn) values versus the cycle
number. The ∆Rn versus cycle Amplification plot is used for calculation threshold
cycle (CT).
analysis session
A set of analysis results derived from a single raw data set associated with a single
plate document analyzed by the SDS software. The analysis session is persisted in the
database and can be retrieved by the SDS software. Multiple analysis sessions can be
created from the same plate document, however each analysis session must have a
unique name. An analysis session does not have to be associated with a Study;
however, if it has such a relationship, it can be associated with at most one Study at
any given time.
annealing
Binding a probe to the DNA or cDNA during the PCR process
Application
Program Interface
(API)
A set of routines, protocols, and tools for building software applications. A good API
makes it easier for a programmer to develop a program by providing all the building
blocks necessary to link the elements of the various subcomponents of the system.
assay type
With reference to the SDS 2.2 Enterprise Option software, an assay type is one of the
following: allelic discrimination, absolute quantification, or relative quantification.
auto increment
value
Method setting that increases or decreases the value for any PCR set-point parameter
(time or temperature) by a fixed amount every cycle.
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background
An inherent spectral component of the signal generated by a fluorescence assay that
is attributed to the Applied Biosystems 7900HT Fast Real-Time PCR System.
Uncorrected background signal can interfere with the sensitivity of the SDS software
and its ability to determine CTs. For this reason background signal is measured during
calibration and is removed mathematically during multicompoenting.
baseline
In terms of the Amplification plot, the baseline is a line fit to defined range of cycles
before the sequence detection system detects the amplification of the PCR product.
For standard PCR assays, the SDS software uses a default range of cycles 3-15 to
establish the baseline for a PCR assay.
calibrator
In relative quantification analysis a sample to which all other samples are referenced
on a detector-by-detector basis.
cDNA
(complementary
DNA)
A DNA molecule with a nucleotide sequence that is complementary to an RNA
molecule. cDNA is made from RNA through the action of the enzyme Reverse
Transcriptase. RNA is converted to cDNA because cDNA is more robust than fragile
RNA, and because DNA templates are required for many applications, including PCR.
clipped data
In real time mode, fluorescent signal intensity is collected for the entire plate every
8.5 seconds. Clipped data points are calculated by averaging the last three computed
Rn (normalized reporter signal) data points collected during the extension phase of
each cycle repetition. Clipped data are presented as baseline uncompensated data
(Rn vs. cycle data) and baseline compensated data (∆Rn vs. cycle data).
command line
Some executable programs can be invoked in a DOS Command Prompt window
and/or from within a.bat script file, to run usually without a graphic user interface
(GUI). Such programs may support user specification of parameters via a command
line, where those parameters would be entered after the name of the executable
program. Often these command line parameters are filenames, directory paths, or flag
settings.
consumable
(1) A vessel used to contain reactions run on the 7900HT instrument (a microplate or
a TaqMan® Low Density Array). (2) A reagent kit, chemical, or disposable labware
used as part of an assay.
container type
The type of plate to be analyzed by the 7900HT instrument. The types are:
ABI PRISM™ 96-Well Optical Reaction Plates, ABI PRISM™ 384-Well Optical
Reaction Plates, Optical 96-Well Fast Thermal Cycling Plates, and TaqMan® Low
Density Arrays.
cycle set-point
(1) A cycle set-point is any temperature desired for a sample block or heated cover.
(2) In the context of thermal cycling, cycle set-point could represent one or more
temperatures in a step or stage.
delta CT
See ∆CT.
delta delta CT
See ∆∆CT.
delta Rn
See ∆Rn.
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detector
A virtual representation of a gene- or allele-specific nucleic acid probe reagent used
for absolute quantification, relative quantification, allelic discrimination, or other
analyses performed on an SDS instrument. Examples of reagents represented as
detectors include TaqMan probes and the SYBR Green 1 dsDNA Binding Dye.
endogenous
control
Detector for a gene presented at a consistent level in all experimental samples. By
using an endogenous control as an active reference, one can normalize quantification
of a messenger RNA (mRNA) target for differences in the amount of total RNA added
to each reaction.
endogenous
reference
See endogenous control.
end-point
(plate read)
run/analysis
Refers to processing of samples for allelic discrimination and plus/minus scoring
chemistries of samples by the 7900HT instrument. The 7900HT instrument collects a
single reading after prior PCR temperature cycles are completed using a thermal
cycler. After analysis by the SDS software, the resulting multi-component data is used
to assess the presence of target sequences in the unknown samples.
expression fold
value
The expression of a target gene relative to control gene in one sample, relative to the
expression level in a calibrator sample. See ∆∆CT.
hands-off
automation
After plates have been loaded into the Zymark® Twister Microplate Handler stacks,
and the Automation Controller Software has been told to start running, no human
intervention should normally be required until all plates in the robot stacks have been
processed. With the SDS Enterprise Database option, this includes plate document
analysis and analysis results persistence to the database.
high throughput
(HT)
Describes the ability to analyze a large quantity of samples for genotyping or gene
expression analysis.
hold
A thermal cycling set-point of a method that maintains a specified temperature for an
extended period of time.
locus
The physical location of a site on a chromosome, sometimes understood as the site of
a gene, usually understood as the site of a marker variant.
marker
For SDS software, a virtual representation of a variable segment of DNA (such as a
SNP) whose inheritance can be followed, comprising a name and a set of two detectors
specific for the allelic variants. Markers are used only for allelic discrimination
assays, not for absolute quantification, or relative quantification. Allelic
discrimination plate documents must contain at least one marker. The marker must be
configured with two detectors before it can be applied to a plate document.
method
The protocol that the Applied Biosystems 7900HT Fast Real-Time PCR System
follows during a real-time or end-point sequence detection run. Methods used by the
7900HT instrument make use of the following information: thermal cycling times and
temperatures, ramp rates, auto-increment values, and data collection settings.
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minor groove
binder (MGB)
One of a class of molecules which form close atomic contacts in the deep, narrow
space formed between the two phosphate-sugar backbones in the DNA double helix.
Conjugation of a minor groove binder to oligonucleotides stabilizes nucleic acid
duplexes causing a dramatic increase in oligonucleotide Tm. Fluorogenic probes with
the MGB attached to the 3′ end perform well in the 5′ nuclease assay. They are an
improvement over unmodified probes because shorter sequences (13- to 20-mers) can
be used to obtain probes that have an optimal Tm. Thus, attachment of the MGB
enables the use of shorter fluorogenic probes, which results in improved mismatch
discrimination.
multicomponenting
The analysis performed by the SDS software used to distinguish the contribution of
individual dyes to the raw spectral data collected by the 7900HT instrument. The
overlapping spectra from the individual dye components in a well generate the
composite spectrum that represents a raw data fluorescent reading.
multi-reporter
(multiplex) PCR
A technique that uses multiple fluorogenic dye-labeled TaqMan® probes to detect the
simultaneous amplification of two or more target nucleic acids within a DNA sample.
No Template
Control (NTC)
A control reaction with all PCR components included except DNA template. After a
detector is applied to a well, each detector must be assigned a task that describes its
purpose. One of the tasks is NTC and is applied to all detectors of negative control
wells containing PCR reagents but lacking samples.
normalized reporter
signal (Rn)
See Rn.
nucleotide
One of the structural components, or building blocks, of DNA and RNA. A nucleotide
consists of a base (one of four chemicals: adenine, thymine, guanine, and cytosine)
plus a molecule of sugar and one of phosphoric acid.
outlier
An unusually extreme value for a variable, given the statistical model in use. In the
SDS software, automatic outlier removal in RQ Studies is conducted to assist in the
elimination of data contamination occurring when a process or phenomenon other
than initial sample quantity affects the measured CT.
PCR master mix
A reagent mix containing all components necessary for PCR except template DNA
and primers/probes. For example, the TaqMan® 2✕ Universal PCR Master Mix
contains AmpliTaq Gold® DNA polymerase, AmpErase® UNG that protects against
carry-over contamination, Passive Reference I for signal normalization in all 5′
nuclease assays, dNTPs and optimized buffer components.
persist(ed)
Stored in, and thus retrievable from, the database used by the SDS software.
pure dye
Pure dye data is generated from the results of a pure dye run in which the SDS
software collects spectral data from a set of dye standards during a 2-min. hold at
60 °C. The software stores the spectral information for the pure dye standards within
a calibration file located in the SDS directory. This calibration file is used for
calculating multi-component data. After the run, the software extracts each
component dye spectrum from the collected data in the pure spectra run file. Because
the age and use of the instrument components can affect the pure spectra readings,
Applied Biosystems recommends updating the pure spectra data files once or twice
annually depending on instrument use.
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quality value
The quality value for a given point is an estimate of the probability that the assigned
genotype for that point is correct, with respect to the rest of the points in the data set.
The term quality value is only used for allelic discrimination autocalling.
quantity
(1) the quantity values for the standards contained on a plate (2) the initial sample
quantity computed for the unknowns contained on a plate.
quantity mean
Statistical mean of the normally distributed quantity values for a replicate group
(wells with the same sample name, detector, and task). Calculated as the sum of all
quantity observations (readings) in the replicate group divided by the number of
replicates.
quantity standard
deviation
Statistical standard deviation of the normally distributed quantity values for a replicate
group (wells with the same sample name, detector, and task). The standard deviation
is calculated using the nonbiased or “n-1” method (square root of sum of the squared
normed residuals divided by n-1 degrees of freedom, with n being the number of
replicates.
quencher dye
The fluorescent dye (or non-fluorescent) banned to the 3′ end of the TaqMan® Probe,
which reduces fluorescence of the reporter dye at the 5′ end by FRET until the reporter
dye is cleared during PCR.
ramp
(temperature ramp)
The interval between two set-points during which the instrument heats or cools the
samples.
raw data
Unmodified spectral data detected between the wavelengths of 500 nm and 660 nm
collected by the 7900HT instrument during a real-time or end-point run.
reaction device
The physical device used to hold the PCR reactions during evaluation on the
Applied Biosystems 7900HT Fast Real-Time PCR System. Examples of reaction
formats include ABI PRISM™ 384- and 96-Well Optical Reaction Plates or the
TaqMan® Low Density Array.
real-time
run/analysis
Refers to real-time PCR processing of individual plates by the 7900HT instrument for
absolute and relative quantification. The 7900HT instrument collects fluorescence
data during each cycle of a pre-programmed PCR run. The pre-programming occurs
by setting the thermal cycling parameters of a default method. The step parameters
(time and temperature) are adjusted, hold, cycle set, or step are selected, and
optionally, auto increment values, ramp rate values and data collection options can be
configured.
relative
quantification
A process by which the quantity of a nucleic acid target, relative to a control target, in
one sample is calculated relative to the quantity in another sample. For example, the
quantity of c-myc in a brain tissue sample relative to the quantity of c-myc in liver
tissue sample.
reporter dye
Fluorescent dye used to bind to the 5′ end of the TaqMan® Probe. There are a number
of dyes, such as FAM™ and VIC® that covalently bind to the 5′ end of the probe.
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Rn
The normalized reporter signal is the cycle-by-cycle ratio of the fluorescence of the
reporter dye and the fluorescence of the passive reference dye in a given well. During
PCR, Rn increases as the amplification copy number increases until the reaction
approaches a plateau. When using the instrument in its Plate Read mode (end point
analysis), the fluorescence signal is read at a single point in time after the completion
of PCR rather than at intervals during the course of PCR.
run
A run describes a collection of fluorescence data carried out by the 7900 instrument
during and/or after cycling of samples in an optical plate or TaqMan® Low Density
Array.
session
See Analysis Session.
single nucleotide
polymorphism
(SNP)
A single base pair of DNA in the genome that differs between individuals.
singleplex
A PCR reaction with a single fluorescent detector per well.
SNP
See single nucleotide polymorphism.
spectral bin
A portion of the complete range of the wave length measured by the 7900HT
instrument (approximately 500-660 nm).
spectral calibration
Spectral calibration occurs from pure dye results obtained from a pure dye run. The
software stores the spectral information for the pure dye standards within a calibration
file located in the SDS directory. After a run, the software extracts each component
dye from the collected data in the pure spectra run file. Because the age and use of the
instrument components can affect pure spectra readings, Applied Biosystems
recommends updating the pure spectra run file once or twice annually depending on
instrument use.
spectral crosstalk
The fluorescent contribution of one dye signal to another caused by the overlap of their
spectra.
stack
The physical container of a 7900HT instrument into which well plates can be stacked.
There are 5 stacks in all. Four of the five consecutively numbered stacks are used to
hold the sample plates while the fifth is used by the Plate Handler to hold the first set
of analyzed plates. Maximally 20 plates can be placed in each stack. The instrument
can process maximally eighty plates in one batch.
study
A study in the SDS Enterprise Database option is a named collection of zero or more
AD or RQ analysis sessions. A study in the SDS software main application is an '.sdm'
plate document file that contains one or more '.sds' relative quantification plate
document files.
task
A setting applied to the detectors within a well of a plate document that determines
how the data collected from the well during analysis are used. The task selections
available differ depending on the plate's assay type.
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template
(1) [SDS software: SDS 7900HT Template Document or plate document template] A
pattern for creating an SDS 7900HT Document (plate document). The filename
extension for a plate document template is *.sdt. The plate document template
facilitates creation of plate documents in the SDS software. It is typically a starting
point for batch document setup. The binary representation of the plate document
template is persisted in the database, allowing the plate document template to be
retrieved and then populated with data and saved as an SDS 7900HT Document.
(2) [Chemical] the DNA sample that is being amplified during PCR.
threshold
An arbitrary level of normalized reporter signal (Rn) for CT determination in real-time
assays. The level is set to be above the baseline and sufficiently low to be within the
exponential growth region of the amplification region of an amplification curve. The
threshold is the line whose intersection with the Amplification plot defines the CT.
threshold cycle (CT)
For a given well, the threshold cycle (CT) represents the PCR cycle at which the SDS
software first detects a noticeable increase in reporter fluorescence above a baseline
signal.
Tm
(1)[Chemical] The temperature at which 50% of the DNA amplicons are in a
double-stranded configuration. (2) [Mathematical] The maximum value for the first
derivative curve of normalized reporter fluorescence (Rn).
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Index
Symbols
– 6-31
↑ 6-33
Numerics
7900HT instrument, See instrument
9600 emulation mode 3-19
A
ABI PRISM 7700 Sequence Detection System,
emulating 3-19
ABI PRISM Sealer, see Sealer
absolute quantification
about 6-6 to 6-7
analyzing data 6-9 to 6-13
assay development guidelines B-6
procedure checklist 6-9
selecting and preparing standards B-6
troubleshooting 8-12
adding
bar code to a plate document 2-27, 3-25
custom dyes to the pure dye set 7-27
detector tasks to a plate document 3-14
detectors to a plate document 3-11
markers to a plate document 3-13
plate documents to the plate queue 4-35, 4-40
sample names to a plate document 3-25
adjusting
analysis options for absolute quantification 6-11
display settings 3-22
method step parameters 3-19
plate-sensor switch 7-37 to 7-40
adjustment knob 7-36
air bubbles 8-6
aligning
fixed-position bar code reader 7-49 to 7-51
Plate Handler 7-41 to 7-48
allele calls
about 5-15
calling 5-14
scrutinizing 5-16
allelic discrimination
about 5-6 to 5-8
analyzing run data 5-10 to 5-18
assay development guidelines B-5
maximizing throughput 3-3
procedure checklist 5-10
thermal cycling on the 7900HT instrument 3-18
troubleshooting 8-13
Allelic Discrimination Plot
about 5-13
calling alleles 5-14
datapoint cluster variations 5-8
exporting as a graphic file A-16
exporting data as a text file A-16
genotypic segregation 5-8
outliers 5-8
scrutinizing allele calls 5-16
amplification
beyond reproducible limits in multi-reporter
experiments 6-19
amplification curve
about D-4
Geometric (Exponential) Phase D-4
Linear Phase D-5
Plateau Phase D-5
Amplification Plot
exporting as a graphic file A-16
exporting data as a text file A-16
Analysis 6-22
analysis sessions, about 1-28
analyzing
absolute quantification data 6-9 to 6-13
allelic discrimination data 5-10 to 5-18
background data 7-19
dissociation curve data 6-40 to 6-44
pure dye data 7-25
relative quantification data 6-22 to 6-35
applying
detector tasks 3-14
detectors to a plate document 3-11
markers to a plate document 3-13
sample names 3-25
architecture
database LAN setup 1-23
database WAN setup 1-24
Archiver 1-26
archiving files 7-54
arrows, sample bars 6-31
assay development guidelines
absolute quantification B-2 to B-4, B-6
allelic discrimination B-2 to B-5
DRAFT
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November 19, 2007 11:09 am, 4351684AIX.fm
Index-1
relative quantification B-6
automatic outlier removal 6-19
Automation Accessory
components 7-36
Plate Handler, See Plate Handler
See fixed-position bar code reader
Automation Controller Software
about 4-39
adding plates to the plate queue 4-40
configuring for operation 4-42
ejecting a plate 4-44
launching 4-39
monitoring instrument progress 4-44
removing plates from the plate queue 4-40
starting the plate queue 4-43
stopping the plate queue 4-44
B
background run 7-16 to 7-19
about 7-16
constructing a background plate 7-17
creating a plate document 7-17
extracting 7-19
preparing a background plate 7-17
troubleshooting 8-9
when to perform 7-16
bar code information
entering into a plate document 2-27, 3-25
bar code readers
fixed position, See fixed-position bar code reader
hand held, See hand-held bar code reader
bars, in the Gene Expression Profile
symbols 6-31
baseline
about D-6
configuring value for automatic analysis 6-11
Block readout (from the Real Time tab) 4-27
Bucket Type, setting on centrifuge 4-13
C
calibrating the 7900HT instrument
adjusting the plate-sensor switch 7-37
aligning the fixed-position bar code reader
aligning the Plate Handler 7-41
performing background run 7-16
performing pure dye (spectral) run 7-20
calibrator sample 6-20
Centrifuge system
bucket requirements 4-16
Bucket Type, setting 4-13
components 4-12
installation and operation 4-13
placing bucket in centrifuge 4-17
requirements 4-12
TaqMan Low Density Array, placing in
Index-2
7-49
DRAFT
bucket 4-16
Centrifuging, TaqMan Low Density Array 4-16
changing
gripper finger pads 7-52
pane, view, and plot sizes 2-21
sample block module 7-6
checklists
absolute quantification 6-9
allelic discrimination 5-10
dissociation curve 6-22, 6-40
relative quantification 6-22
chemistry
5´ Nuclease (TaqMan) D-2
non-optimized 8-6
SYBR Green D-3
troubleshooting 8-5 to 8-8
cleaning
gripper finger pads 7-52
sample block wells 7-15
closing
instrument tray (Automation Controller
Software) 4-44
instrument tray (SDS software) 4-29
plate documents 2-18
Zymark Twister Software 7-48
comments
adding to a detector 3-9
adding to a plate document 3-25
Comparative CT Method
about 6-3, D-8
formula derivation D-8
Components
centrifuge system 4-12
TaqMan Low Density Array 4-10
computer
about 1-6
hard drive partitions 1-6
maintaining 7-54
minimum system requirements 1-6
troubleshooting 8-14
turning ON 2-4
configuring the 7900HT instrument for 9600
emulation 3-19
consumables
384-Well Optical Reaction Plates C-3
96-Well Optical Reaction Plates C-4
improper or damaged plastics 8-8
Optical 96-Well Fast Thermal Cycling Plates C-4
Optical Adhesive Covers C-3
Optical Cap Strips C-3
writing on reaction plates 8-7
Contamination
genomic DNA 4-11
preventing 4-6
contamination
decontaminating the sample block 7-14
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, 4351684AIX.fm
fluorescent, common sources 8-7
isolating on the sample block module 8-9
contextual menus, using 2-28
Control assay 4-11
copying
detectors to a plate document 3-11
markers to a plate document 3-13
Cover readout (from the Real Time tab) 4-27
creating
background plate documents 7-17
custom pure dye plate documents 7-28
detectors 3-9
markers 3-12
plate documents 2-13, 3-8
plate documents from a template 3-24, 4-36
pure dye plate documents 7-22
CT, See threshold cycle
custom pure dyes
adding to the pure dye set 7-27 to 7-29
creating a custom pure dye plate 7-27
cycle set, adding to a method 3-19
D
data type definitions (exportable) A-17
database
saving plate documents 4-24
saving results 5-19, 6-53
saving templates 3-23
database connection A-22
decontaminating the sample block module 7-14 to 7-15
defragmenting the hard drive 7-54
deleting, steps from a method 3-19
detector tasks
about 3-14
applying 3-14
importing into a plate document A-2
detectors
about 3-9
applying to a plate document 3-11
copying to a plate document 3-11
creating 3-9
importing into a plate document A-2
tasks, See detector tasks
determining melting temperatures 6-44
diagram
database LAN setup 1-23
database WAN setup 1-24
disconnecting the SDS software 4-30
display settings, adjusting 3-22
dissociation curve
about 6-38
analyzing 6-40 to 6-44
definitions of the Tm value 6-43
procedure checklist 6-22, 6-40
programming a temperature ramp 3-21
Dissociation Plot
about 6-42
exporting as a graphic file A-16
exporting data as a text file A-16
E
ejecting a plate from the 7900HT instrument 4-29,
4-44
endogenous control 6-20
end-point runs
about 5-2
allelic discrimination, See allelic discrimination
entering
bar code information 2-27, 3-25, 4-36
comments 3-9, 3-25
sample names into a plate document 3-25
Equipment needed 4-12
exportable data type definitions A-17
exporting
data from a plate document 2-14, A-16
plots and views as graphic files A-16
External endogenous control assay 4-11
F
finger pads
cleaning 7-52
replacing 7-52
fixed-position bar code reader
aligning 7-49 to 7-51
connections 1-10
location 1-7
specification 1-7
fluorescent
contamination 8-7
detection system 1-10
fluorogenic probe
about D-2
designing B-2 to B-3
G
gene expression level
maximum 6-33
minimum 6-33
gene expression levels 6-31
Gene Expression Plot 6-31
Genomic DNA contamination, minimizing
genotypic segregation of datapoints
(Allelic Discrimination Plot) 5-8
Geometric (Exponential) Phase D-4
graph, Gene Expression Profile
sample bars 6-31
X-axis 6-31
4-11
DRAFT
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November 19, 2007 11:09 am, 4351684AIX.fm
Index-3
Y-axis 6-31
grey dividing line, using 2-21
grid, See plate grid
gripper 7-36
guidelines
absolute quantification assay development B-6
allelic discrimination assay development B-5
loading the Plate Handler 4-41
relative quantification assay development B-6
TaqMan probe design B-2 to B-3
IP Address A-22
irreproducibility
causes 8-5 to 8-8
H
L
hand-held bar code reader
connections 1-10
location 1-7
specification 1-7
using 2-27
hard drive
defragmenting 7-54
partitions 1-6
heated clamp 1-4
help
background information 2-3
using the Sequence Detection Systems Software
Online Help 2-3
hold, adding to a method 3-19
hotkey combinations, using 2-28
launching
Automation Controller Software 4-39
SDS software 2-7
LAVA software
about 1-14
aligning the fixed-position bar code reader
7-51
launching 7-49
LDHost software, See LAVA software
learning to use the SDS software 2-10
lights, See instrument, status lights
limited warranty statement E-1
Linear Phase of the amplification curve D-5
loading plates
into the instrument 4-25
into the Plate Handler stacks 4-42
low copy templates 8-8
I
importing setup table data 2-16, A-2
imprecise pipetting 8-6
improper threshold setting 8-5
installing
plate adapter 7-12
sample block module 7-6
SDS software 7-55
the operating system software 7-55
instrument 1-4
about 1-2 to 1-10
external components 1-3
firmware 1-14
internal components 1-4
loading plates 4-25
maintaining 7-3
optics system 1-5
status lights 2-5
supported runs and chemistries 1-2
troubleshooting 8-14
turning ON 2-4
instrument tray 4-44
opening and closing (Automation Controller
Software) 4-44
opening and closing (SDS software) 4-29
replacing the plate adapter 7-12
Index-4
DRAFT
J
Java Runtime Environment, about
1-14
K
keyboard shortcuts, using
2-28
7-49 to
M
maintenance schedule 7-3
managing SDS data 1-17
markers
about 3-12
applying to a plate document 3-13
copying to a plate document 3-13
creating 3-12
importing into a plate document A-2
master mixes
preparing B-3
using 8-6
maximizing, instrument throughput 3-3
maximizing/minimizing panes, views, and plots 2-22
maximum
gene expression level 6-33
melting temperature
definition 6-43
determining 6-44
methods
about 3-17 to 3-21
adding a hold, cycle set, or step 3-19
adding a temperature ramp 3-21
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, 4351684AIX.fm
adjusting step parameters 3-19
configuring data collection options 3-20
programming 3-18 to 3-21
removing a step 3-19
setting the sample volume 3-20
minimum
gene expression level 6-33
modes of operation 1-17
automated 1-18
database 1-18
stand-alone 1-17
mRNA targets, number per well 4-10
N
no template control (NTC)
detector task 3-14
NTC calls applying 5-15
NTC calls verifying 5-16
Number of targets per well 4-10
O
↓ 6-33
opening
instrument tray (Automation Controller
Software) 4-44
instrument tray (SDS software) 4-29
plate documents 2-19
operating system
supported 1-6
upgrading 7-55
operating the 7900HT instrument
power switch 2-4
optimizing primer and probe concentrations
outliers
allelic discrimination 5-8
automatic outlier removal 6-19
outlying amplification
multi-reporter experiments 6-19
P
panes
hiding 2-21
maximizing/minimizing 2-22
resizing 2-21, 2-22
showing 2-21
passive reference
setting 3-16
PCR
5´ Nuclease Assay D-2
Geometric (Exponential) Phase
kinetic analysis of D-4
Linear Phase D-5
Plateau Phase D-5
SYBR Green Chemistry D-3
D-4
B-3
PCR step
fill reservoirs, checking after centrifuging 4-18
fill reservoirs, trimming after sealing 4-21
general practices 4-6
placing in bucket in centrifuge 4-17
TaqMan Low Density Array, centrifuging 4-16
TaqMan Low Density Array, loading 4-14
TaqMan Low Density Array, placing in
bucket 4-16
TaqMan Low Density Array, sealing 4-19
persisted data
analysis sessions 1-28
plate documents
1-28
studies 1-28
pipetting errors 8-6
pipettors, using 8-6
plate adapter, changing 7-12
plate document information
applying to a plate document 3-25
plate documents 3-8
about 2-12
adding to the plate queue 4-35, 4-40
applying detector tasks 3-14
applying sample names 3-25
assigning standard quantities 3-15
closing 2-18
configuring document information 3-25
copying detectors 3-11
copying markers 3-13
creating 2-13, 3-8
creating from a template 3-24, 4-36
exporting 2-14
importing setup data 2-16, A-2
methods, See methods
opening 2-19
programming methods 3-17
removing from the plate queue 4-40
running batches 4-39
running individually 3-26
saving 2-17
saving as a single plate file 4-24
saving as a template file 3-22
saving to the database 4-24
setting Sample Volume 3-20
setting the passive reference 3-16
plate grid
about 2-29
selecting wells 2-24
viewing well information 2-23
zooming 2-26
Plate Handler 1-8, 7-36
adjustment knob 7-36
aligning 7-41 to 7-48
cleaning the finger pads 7-52
gripper 7-36
plate stack positions 7-36
DRAFT
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November 19, 2007 11:09 am, 4351684AIX.fm
Index-5
plate-sensor switch 7-36
replacing the finger pads 7-52
turning ON 2-4
plate queue
adding plate documents (Automation Controller
Software) 4-40
adding plate documents (SDS software) 4-35
adding plate documents (Template Batch
utility) 4-36
removing plate documents 4-40
starting 4-43
stopping 4-44
plate stacks
loading plates 4-41, 4-42
placing in use 4-42
positions 7-36
Plateau Phase of the amplification curve D-5
plates
running batches 4-39, 4-41
running individually 4-25
types, See consumables
plate-sensor switch 7-36
adjusting 7-37 to 7-40
plots
hiding 2-21
maximizing/minimizing 2-22
resizing 2-21, 2-22
showing 2-21
Port Number A-22
Post readout (from the Plate Read tab) 4-27
Pre readout (from the Plate Read tab) 4-27
precision
causes of low precision 8-5 to 8-8
preparing
master mixes B-3
plate for a run 4-6
primer and probe concentrations
optimizing B-3
programming
methods (absolute quantification) 3-19
methods (allelic discrimination) 3-18
methods (dissociation curve analysis) 3-21
methods (relative quantification) 3-19
temperature ramp 3-21
Protocol
preventing contamination 4-6
pure dye plate
constructing for custom dyes 7-27
preparing for use 7-24
pure dye run
about 7-20
creating a plate document 7-21
extracting data 7-25
performing 7-24
troubleshooting 8-11
when to perform 7-20
Index-6
DRAFT
Q
Quality control, TaqMan Low Density Array 4-12
quantifying
probes and primers B-3
standards for absolute quantification B-6
quantitative RT-PCR
about 6-3
absolute, See absolute quantification
relative, See relative quantification
types of 6-3
quantities, applying to a plate document 3-15
R
R2 readout (from the Standard Curve Plot) 6-13
reagents
custom oligonucleotides C-6
non-Applied Biosystems PCR reagents 8-8
TaqMan Pre-Developed Assays and Reagents C-6
TaqMan RNase P Instrument Verification
Plates C-5
re-connecting the SDS software 4-30
relative quantification
about 6-3, 6-16 to 6-20
analyzing data 6-22 to 6-35
assay development guidelines B-6
procedure checklist 6-22
the Comparative CT Method 6-3
the Standard Curve Method 6-3
troubleshooting 8-12
removing
plate documents from the plate queue 4-40
steps from a method 3-19
Rep readout (from the Real Time tab) 4-27
replacing
gripper finger pads 7-52
sample block module 7-6
replicate wells, use of 6-20
reproducible limits 6-19
resizing panes, views, and plots 2-21
restacking plates 4-42
restoring
database connection A-22
RQ Manager Software 1-25
RQ Min/Max Confidence 6-28
running
batches of plates 4-39, 4-41
single plate 4-25
S
sample
calibrator 6-20
sample bars
about 6-31
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, 4351684AIX.fm
sample block locking bar 7-8
sample block locking bolt 7-8
sample block module
about 1-5
cleaning sample block module wells 7-15
contamination 8-7
replacing 7-6
sample names
adding to a plate document 3-25
importing into a plate document A-2
Sample readout (from the Real Time tab) 4-27
sample volume setting 3-20
saturation, signal 7-27
saving
plate documents 2-17, 4-24
template files 3-22
scrutinizing allele calls 5-16
SDS 7900HT Document (*.sds) files
See plate documents
SDS 7900HT Template Document (*.sdt) files
See templates
SDS Enterprise Database
about 1-22
command line tools 1-26
RQ Manager Software 1-25
saving plate documents 4-24
saving results 5-19, 6-53
saving templates 3-23
SDS Database 1-26
SDS User Account Manager 1-27
SNP Manager Software 1-25
study-centric design 1-27
SDS software
about 1-14
disconnecting 4-30
installing 7-55
launching 2-7
learning to use the software 2-10
re-connecting 4-30
upgrading 7-55
Sealer
installation 4-14
overview 4-14
Sealer, see microfluidic card sealer
Sealing
guidelines 4-19
TaqMan Low Density Array 4-19
trimming fill reservoirs after sealing 4-21
selecting
wells from the plate grid 2-24
Service Name A-22
setup table file
about A-4
example files A-4
exporting A-17
importing into a plate document A-2
structure A-4
signal saturation 7-27
Slope readout (from the Standard Curve Plot) 6-13
SNP Manager Software 1-25
spectral calibration, See pure dye runs
Stage readout (from the Real Time tab) 4-27
Standard Curve Method 6-3
Standard Curve Plot
about 6-13
exporting as a graphic file A-16
exporting data as a text file A-16
standards
quantifying for absolute quantification B-6
selecting for absolute quantification B-6
selecting for relative quantification B-6
starting
plate queue 4-43
run from the SDS software 4-25
State readout (from the Real Time tab) 4-27
Status readout (from the Real Time tab) 4-27
step
adding to a method 3-19
Step readout (from the Real Time tab) 4-27
stopping
plate queue 4-44
run from the SDS software 4-28
studies 1-28
study-centric design 1-27
SYBR Green 1 Dye
about D-3
System performance, verifying
RNase P card, preparing 7-32
T
table pane
about 2-29
exporting A-16
TaqMan fluorogenic probe
about D-2
designing B-2 to B-3
TaqMan Low Density Array
centrifuging 4-16
components 4-10
handling 4-14
how it works 4-10
loading 4-14
loading guidelines 4-14
number of targets per well 4-10
overview 4-10
placing in bucket 4-16
quality control 4-12
removing from packaging 4-14
sealing 4-19
DRAFT
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, 4351684AIX.fm
Index-7
single-plex TaqMan reagents 4-11
TaqMan primers and probes 4-10
trimming after sealing 4-21
TaqMan reagents, description 4-10
TaqMan RNase P Instrument Verification Plates
about 7-30
analyzing 7-34
kits C-5
preparing a plate document 7-33
running 7-30, 7-32
Targets, mRNA, number per well 4-10
tasks, See detector tasks
templates
about 2-12
creating a single plate document 3-24
creating multiple plate documents 4-36
saving as 3-22
thermal cycler block, See sample block module
thermal cycling protocol, See methods
threshold
about D-6
configuring for automatic analysis 6-11
improper setting 8-5
threshold cycle
calculation D-6
relationship to PCR product D-7
Time readout (from the Real Time tab) 4-27
Time Remaining readout (from the Real Time
tab) 4-27
Tm, See melting temperature
troubleshooting 8-2 to 8-20
7900HT instrument 8-14
background runs 8-9
chemistry problems 8-5 to 8-8
computer 8-14
end-point runs 8-13
fixed-position bar code reader 8-17, 8-20
pure dye runs 8-11
real-time runs 8-12
SDS software 8-14
Zymark Twister Microplate Handler 8-17, 8-20
turning ON the Applied Biosystems 7900HT Fast RealTime PCR System 2-4
resizing 2-21, 2-22
showing 2-21
W
warranty statement E-1
well inspector 2-31
Y
Y Inter readout (from the Standard Curve Plot)
6-13
Z
zooming
allelic discrimination plot 5-14
plate grid wells 2-26
Zymark Twister Microplate Handler
See Plate Handler
Zymark Twister Software
about 1-14
aligning the Plate Handler 7-41 to 7-48
closing 7-48
launching 7-41
testing the plate sensor switch 7-39
U
upgrading
operating system software 7-55
SDS software 7-55
V
viewing, well information 2-23
views
hiding 2-21
maximizing/minimizing 2-22
Index-8
DRAFT
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
November 19, 2007 11:09 am, 4351684AIX.fm
Worldwide Sales and Support
Applied Biosystems vast distribution and
service network, composed of highly trained
support and applications personnel,
reaches 150 countries on six continents.
For sales office locations and technical support,
please call our local office or refer to our
Web site at www.appliedbiosystems.com.
Applied Biosystems is committed to
providing the world’s leading technology
and information for life scientists.
Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
06/2010
www.appliedbiosystems.com
Part Number 4351684 Rev. C
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