QuantiGene 2.0 miRNA Assay User Manual

QuantiGene 2.0 miRNA Assay User Manual

User Manual

QuantiGene

®

2.0 miRNA Assay

P/N 13114 Rev.A 110331

For research use only.

Not for use in diagnostic procedures.

Trademarks

Affymetrix ® and are trademarks of Affymetrix, Inc.

QuantiGene is a registered trademark exclusively licensed to Affymetrix, Inc.

Affymetrix®, Axiom™, Command Console®, DMET™, GeneAtlas™, GeneChip®, GeneChip-compatible™, GeneTitan®,

Genotyping Console™, myDesign™, NetAffx®, OncoScan™, and Powered by Affymetrix™ are trademarks or registered trademarks of Affymetrix, Inc. Luminex® and xMAP® are registered trademarks of Luminex Corp.

All other trademarks are the property of their respective owners.

Limited License

Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a nonexclusive, non- transferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix. You understand and agree that, except as expressly set forth in the

Affymetrix terms and conditions, no right or license to any patent or other intellectual property owned or licensable by

Affymetrix is conveyed or implied by this Affymetrix product. In particular, no right or license is conveyed or implied to use this

Affymetrix product in combination with a product not provided, licensed, or specifically recommended by Affymetrix for such use.

Patents

Arrays: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334;

6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; 6,955,915 and D430,024 and other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637 and 6,054,270.

Software products may be covered by one or more of the following patents: U.S. Patent Nos. 6,090,555; 6,611,767;

6,687,692;6,829,376; 7,130,458; 7,451,047; and other U.S. or foreign patents

Fluidics stations: Products may be covered by U.S. Patent Nos. 6,114,122; 6,287,850; 6,391,623; 6,422,249; and other U.S. or foreign patents. Scanners: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,141,096;

6,262,838; 6,294,327; 6,403,320; 6,407,858; 6,597,000; 7,406,391; and other U.S. or foreign patents.

Hybridization ovens: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,050,719; 6,386,749;

6,705,754; and other U.S. or foreign patents.

Citing QuantiGene 2.0 miRNA Assay in Publications

When describing a procedure for publication using this product, please refer to it as the QuantiGene 2.0 miRNA Assay.

Disclaimer

Affymetrix, Inc. reserves the right to change its products and services at any time to incorporate technological developments.

This manual is subject to change without notice.

Although this manual has been prepared with every precaution to ensure accuracy, Affymetrix, Inc. assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information.

Copyright

© 2011 Affymetrix Inc. All rights reserved.

Contents

Chapter 1

Chapter 2

Chapter 3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

About This User Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Who This Manual is For . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

What this Manual Covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Contacting Affymetrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Technical Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

QuantiGene 2.0 miRNA Assay Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

How it Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Assay Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

QuantiGene 2.0 Assay Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

QuantiGene 2.0 miRNA Assay Accessory Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

(Optional) Upstream Cell Viability Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Required Materials Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Assay Terminology and Guidelines for Data Analysis . . . . . . . . . . . . . . . . . . 7

Assay Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Assay Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Replicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Assay Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Relative Luminescent Unit (RLU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Assay Limit of Detection (LOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Limit of Quantification (LOQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Assay Linearity/Accuracy of Fold Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Guidelines for Assay Optimization and Assay Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Optimizing Lysis Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Optimizing Sample Input for QuantiGene miRNA Assay . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Assay Replicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Recommended Assay Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Guidelines for Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Normalizing Gene Expression Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Calculating Relative Fold Change of miRNA Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Calculating miRNA Copy Number/Well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

% of Spike Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

QuantiGene 2.0 miRNA Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Assay Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Capturing Target miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Capturing Target miRNA from Cultured Cell or Whole Blood/PAXgene Blood . . . . . . . . 17

Capturing Target miRNA from Fresh, Frozen or FFPE Tissue Homogenates . . . . . . . . . . . 19

Capturing Target miRNA from Total RNA or Purified miRNA. . . . . . . . . . . . . . . . . . . . . . 21

Signal Amplification and Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Preparing Wash Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

iv QuantiGene® 2.0 Reagent System User Manual

Chapter 4

Hybridizing the 2.0 PreAmplifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Hybridizing the 2.0 Amplifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Hybridizing the Label Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Adding the 2.0 Substrate and Detecting Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Low Assay Signal or Poor Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Non-Uniform Signal Across the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

High Background Signal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Well-To-Well Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Day-To-Day Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Appendix A Alternative Plate Washing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Automated Washing Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Appendix B Capture Plate Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

About Capture Plate Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Appendix C Blank Plate Map. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

1

Introduction

About This User Manual

Who This

Manual is For

This manual is for anyone who has purchased QuantiGene 2.0 Assay Kits to perform the

QuantiGene 2.0 miRNA Assay for any of the following sample types:

Cultured cells

Whole blood/PAXgene blood

Fresh, frozen, or formalin-fixed, paraffin-embedded (FFPE) tissues

Total RNA or purified miRNA

What this

Manual Covers

This manual provides:

Background information about QuantiGene 2.0 miRNA Assay and how it works

Experimental design and data analysis

QuantiGene 2.0 miRNA Assay

Troubleshooting

Contacting Affymetrix

Technical Help

For technical support, contact the appropriate resource provided below based on your geographical location. For an updated list of FAQs and product support literature, visit our website at www.affymetrix.com/panomics

.

Table 1.1 Contacting Affymetrix

Location

North America

Europe

Asia

Contact Information

1.877.726.6642 option 1, then option 2 [email protected]

+44 1 628-552550 [email protected]

+81 3 6430 430 [email protected]

2 QuantiGene ® 2.0 miRNA Assay User Manual

QuantiGene 2.0 miRNA Assay Basics

The QuantiGene 2.0 miRNA Assay enables researchers to perform direct detection and quantification of miRNAs at single-base resolution, avoiding biases associated with miRNA or total RNA isolation, cDNA synthesis and PCR amplification. The assay uses validated probe sets that are highly specific for the mature miRNA and do not detect the precursor miRNA. The assay is ideal for target quantification, screening and validation of miRNA array results.

The kits provide the reagents needed to quantify specific miRNA molecules directly from:

Cultured cell lysates

Whole blood or PAXgene blood lysates

Fresh, frozen, or FFPE tissue homogenates

Purified miRNA or total RNA

Please refer to the QuantiGene Sample Processing Kit Package Inserts for instructions on preparing cultured cell or blood lysates or tissue homogenates. To prepare miRNA or total

RNA, follow standard laboratory methods.

The QuantiGene 2.0 miRNA Assay is a hybridization-based assay performed on 96-well plates. The assay is based on direct from lysate quantification of the miRNA target using novel oligonucleotide chemistry and probe design for specific and efficient capturing of miRNA followed by branched DNA (bDNA) signal amplification.

On the first day, the sample is lysed to release the miRNAs and incubated overnight in the

96-well plates with the target specific probe sets (Capture Extenders – CEs and Label

Extenders – LEs).

On the second day the signal amplification tree is built via sequential hybridization of

PreAmplifier (PreAmp), Amplifier (Amp) and Alkaline phosphatase label probe (AP-LP).

The signal is detected by adding chemiluminescent substrate and using a microplate luminometer for the read out.

How it Works

*

*Lumigen ® APS-5Capture

Assay Specifications

Table 1.2 Assay Specifications

Component Description

Assay Format

Sample Types

96 well plate

Purified miRNA

Purified Total RNA

Whole Blood/PAXgene Blood Lysates

Cell Culture Lysates

Plant, Animal and Insect Tissue homogenates

Limit of Detection (Greater than Average Background

Signal + 3SD)

3,000 ~ 60,000 copies/well

Linear range (Entire range must meet accuracy of fold change requirement. Linear regression coefficient {R 2 } greater than 0.98.)

3 Log

% of Spike Recovery

Accuracy of Fold Change (% of Observed/Expected Fold

Change)

100

± 20%, range (80 – 120%)

100

± 20%, range (80 – 120%)

Inter-wells Precision (SD of 3 replicates)

Inter-plates Precision (SD of 3 replicates)

± 10%

± 15%

3

4 QuantiGene ® 2.0 miRNA Assay User Manual

Required Materials

The QuantiGene 2.0 miRNA Assay is a modular kit. Each module below is sold separately in multiple sizes.

QuantiGene 2.0 Assay Kit

QuantiGene Sample Processing Kit (not required for purified RNA samples)

QuantiGene miRNA Probe Set(s)

QuantiGene 2.0

Assay Kit

Components

The components of the QuantiGene 2.0 Assay Kit and their recommended storage conditions are listed below. The QuantiGene 2.0 Assay Kit is available in 4 sizes. Refer to the package insert for quantities of individual components supplied. Kits have a shelf life of 6 months from date of receipt (when stored as recommended) and contain the following components:

Table 1.3 QuantiGene 2.0 Assay Kit Components and Their Storage Conditions

Component Description Storage

2.0 PreAmplifier (PreAmp 1) DNA in aqueous buffered solution

–20 °C

–20 °C 2.0 Amplifier (Amp 1)

Blocking Reagent

DNA in aqueous buffered solution

Aqueous buffered solution containing a preservative

Capture Plate

Label Probe

96-well polystyrene plate coated with capture probes

Oligonucleotide-alkaline phosphatase conjugate in aqueous buffered solution

2.0 Substrate a

Chemiluminescent substrate

Amplifier/Label Probe Diluent Aqueous buffered solution with a protein-containing preservative

Lysis Mixture Aqueous buffered solution containing a preservative

Plate Seals

Wash Buffer Component 1

(Wash Comp 1)

Wash Buffer Component 2

(Wash Comp 2)

Adhesive-backed foil seal

Aqueous solution

Aqueous buffered solution

–20 °C

2–8 °C

2–8 °C

2–8 °C

15–30 °C

15–30 °C

15–30 °C

15–30 °C

15–30 °C

a Lumigen® APS-5

5

QuantiGene 2.0

miRNA Assay

Accessory

Reagents

In addition to QuantiGene 2.0 Assay Kits, two accessory reagents are required to perform

QuantiGene 2.0 miRNA assays.

Table 1.4 QuantiGene 2.0 miRNA Assay Accessory Components

Accessory Reagent Description

Required QuantiGene

Sample Processing Kit

Contains reagents and instructions for processing different sample types: cultured cells, whole blood/PAXgene blood, fresh or frozen tissues or FFPE samples. Specify sample type when ordering (not required for purified RNA).

Required QuantiGene miRNA target-specific Probe

Set(s)

Customer specified miRNA target(s). Each Probe Set contains 1 CE and 1 LE.

Optional QuantiGene miRNA Probe Set(s) for

Normalization

Optional miRNA Positive

Control (250 pMol, 150 million copies/μl)

Customer specified small RNA(s) or miRNA targets suitable for normalizing. These targets should have characteristics of a traditional housekeeping gene. Target is stably expressed under all experimental conditions evaluated. Each Probe Set contains 1 CE and 1 LE.

Synthetic RNA oligo with exactly the same sequence as the target miRNA can be used as an assay positive control or for accurate determination of miRNA copies in samples.

(Optional)

Upstream Cell

Viability Assay

For ordering information, please visit our website at www.affymetrix.com/panomics

The QuantiGene Cell Viability Reagent provides a simple, reliable and sensitive means for quantifying cell proliferation and viability upstream of QuantiGene, QuantiGene Plex and

QuantiGene ViewRNA Plate-Based assays. This homogeneous assay utilizes the nonfluorescent redox dye resazurin, which is converted by metabolically active cells, to resorufin, a highly fluorescent product (Ex 530 – 570 nm; Em 590 – 620 nm).

Table 1.5 QuantiGene Cell Viability Reagent

Component Description

QuantiGene Cell Viability Reagent Non-fluorescent redox dye in aqueous solution

Storage

–20 °C

Required Materials

Not Provided

Other materials required to perform the QuantiGene 2.0 miRNA Assay that are not included in the assay kit are listed here.

Table 1.6 Required Materials Not Provided

Required Material

Adjustable single- and multi-channel precision pipettes for dispensing 1 – 20 μL,

20 – 200 μL and 200 – 1000 μL

Reagent reservoirs:

25-mL capacity

100-mL capacity

Microcentrifuge

Microplate centrifuge that can achieve

240 x g

Vortex mixer

Source

Major laboratory supplier (MLS)

VistaLab

Technologies

Eppendorf

Eppendorf

MLS

Part Number or Model

(P/N 3054-1002) or equivalent

Corning Costar (P/N CLS

4873) or equivalent

541D or equivalent

5804R and rotor A-2 DWP or equivalent

6 QuantiGene ® 2.0 miRNA Assay User Manual

Table 1.6 Required Materials Not Provided

Required Material

Nuclease-free water

Yeast tRNA

Source

MLS

Invitrogen

Luminescence detector with the following features:

Sensitivity > 3 x 10 -21 moles of luciferase

Dynamic range > 8 logs

Well-to-well uniformity

± 5%

Cross-talk < 5 x 10 -5

Fluorescent detection module (optional for

DNA stain); Ex 480 nm/Em 520 nm

Turner BioSystems

Molecular Devices

NOTE:

Make sure your luminometer meets or exceeds minimum performance specifications.

Part Number or Model

P/N 15401-011

Modulus Microplate

Luminometer P/N 9300-001

LMAX or equivalent

QS0700, QS0701 (120V)

QS0710, QS0711 (220V)

Incubator or oven with the following specifications:

Maintain a constant temperatures of

46

± 1 °C

Temperature does not vary more than

± 1 °C

Uniform temperature throughout entire incubator

Affymetrix

4 inch soft rubber roller or

QuantiGene CTC Plate Sealer

Affymetrix

Affymetrix

QuantiGene Incubator Temperature

Validation Kit

NOTE:

Highly recommended for temperature validation of incubator every 6 months.

Affymetrix

Optional. Plate washer that meets or exceeds the following specifications:

30 – 200 μL

± 5% volume

96 or 384 channels

Angle-dispensing tip

Plate stacker

Automation capable

Minimal dead volume

BioTek

QS0515

QG0400

QS0517

ELx 405 model with high throughput pump option

2

Assay Terminology and Guidelines for Data Analysis

Assay Terminology

Assay

Background

A sample well contains all assay components except for target miRNA or sample. The background control is used to determine Limit of Detection (LOD).

Replicates

Technical replicates are replicate assays from a single sample. For example, a cell lysate that is divided into several portions and each portion run in the same assay.

Biological replicates are replicate assays from biologically-equivalent samples. For example, cells grown in different wells that are subjected to the same treatment, lysed independently, then run as distinct samples in the assay.

NOTE: We recommend running 3 technical replicates of each distinct biological sample.

Assay

Precision

The Coefficient of Variation (CV) is a measure of assay precision. QuantiGene 2.0 miRNA

Assay CVs are typically less than 10% for technical replicates.

To determine the assay CV:

Step Action

1

Run technical replicates (n=3) of each sample.

2

3

Calculate the average signal (AVG) of technical replicates from each target miRNA.

Calculate the standard deviation (SD) of signals from technical replicates for each target miRNA.

4

Calculate the %CV.

%CV = (SD/AVG)*100.

Relative

Luminescent

Unit (RLU)

Assay Limit of

Detection (LOD)

The output signal on the luminometer.

Limit of

Quantification

(LOQ)

The LOD is the signal above the background plus 3 standard deviations of the background:

To calculate assay limit of detection for each target miRNA:

LOD = AVG RLU of assay background control wells + 3X SD of assay background signals.

Assay signals below LOD should not be used for quantifications.

LOQ is the lowest RLU that exhibits acceptable accuracy of fold change (see

Assay

Linearity/Accuracy of Fold Change

below).

8 QuantiGene ® 2.0 miRNA Assay User Manual

Assay Linearity/

Accuracy of Fold

Change

Assay linearity is defined as all dilutions that exhibit an accuracy of fold change between

80 and 120%. Assay must exhibit 3 log linearity based on 80 – 120% accuracy of fold change. Typically, the

R 2

(linear regression coefficient) value is greater than 0.98.

To determine assay linearity:

Step Action

1

2

3

Run a dilution series of your sample.

Subtract the AVG assay background signal from the AVG signal of technical replicates for each target miRNA.

Calculate the ratio of background-subtracted AVG RLU from sequential sample dilutions for each target miRNA (80 – 120%).

NOTE:

Quantifiable signals are those signals within the assay’s linear range.

Ratio of Background-Subtracted AVG RLU for Each Target miRNA

3-fold serial dilution of the cell lysate (μL)

Signal

(background subtracted)

(RLU)

Observed fold change

Expected fold change

% Obs/

Exp

60

20

6.6

3,100

1,000

370

3.1

2.70

3

3

103

90

9

Guidelines for Assay Optimization and Assay Design

Overview

Here we provide information and guidelines for:

Optimizing sample lysis

Optimizing sample input

Assay controls

Assay replicates

Calculations of miRNA copy number (relative and absolute)

Optimizing

Lysis Conditions

To determine optimal sample amount for lysis or homogenization:

Step Action

1

Follow the recommended amount of cell number or tissue amount per volume of lysis mixture solution or homogenization solution listed in the Sample Processing

Kit package insert for the specific sample types. Recommendations are summarized below. To ensure optimal lysis, in the initial experiment, run a test range as indicated in the table.

Recommended Sample Preparation Amount

Cultured Cells Tissue

Recommended 400 cells/μL of Working

Lysis Mixture

Test Range 200, 400, 800 cells/μL of

Working Lysis Mixture

5 mg/300 μL of Working

Tissue Homogenization

Solution

2.5, 5.0, 10 mg/300 μL of

Working Tissue

Homogenization

Solution

2

3

For each lysate, prepare a 3-fold serial dilution to determine the assay performance. Assay performance is determined by calculating the following.

LOD

LOQ

Assay linearity

% assay CV

Calculate the assay performance for each sample to determine which one had the best performance and use that amount of cells or tissue for future experiments.

Incomplete or poor lysis will produce high assay CV, poor linearity, and poor LOD.

To determine the optimal lysis method for a sample type:

Following the procedure for determining optimal lysis, test different lysis methods, for example, Tissue lyser or liquid nitrogen for plant tissues

Optimizing

Sample Input for

QuantiGene miRNA

Assay

After you have determined the optimal lysis conditions for sample preparation, use the following guidelines to determine the optimal sample amount/well to use for the

QuantiGene 2.0 miRNA assay:

Resulting signal from the sample is above the LOQ

Amount of sample is high enough to compensate for sample loading error. For example, if the amount of loaded sample can deviate more than 4 times, then increase the sample input by 4 to ensure detection.

10 QuantiGene ® 2.0 miRNA Assay User Manual

If the amount of sample is not limiting, use an input that has a signal/background ratio of at least 3-fold. Background is defined as signal from a sample well without sample input.

Assay Replicates

Run all assay samples with a minimum of duplicates and ideally triplicates. Technical replicates are used to calculate assay precision or %CV.

Recommended

Assay Controls

All experiments should have the following controls:

Assay Background Control. A sample well that contains all assay components except for the target miRNA. The background control is also used to determine the limit of detection (LOD). Data below the LOD should not be used for quantification.

miRNA Positive Control. miRNA positive controls are provided by Affymetrix to be used as the assay positive control. It is also provided to determine the absolute number of miRNA copies/sample/well. The miRNA copy number can be determined by running an 8-point standard curve and linear curve fitting (see data analysis guideline below).

Using the absolute copy number improves data accuracy and precision for inter-day and inter-site experiments and is critical for biomarker discovery and clinical research.

Normalization small RNA(s). Considerations for the selection of the normalization gene to determine relative fold changes or normalize gene expression data across samples or experiments are listed below:

Genes are stably expressed under all experimental conditions evaluated.

Relative luminescent signal should be similar to test samples so no dilution factor is required. In situations where samples are limited, select the normalization gene that is highly expressed and which would require only small amount of sample for detection.

Examples of recommended small RNAs include:

Human

Mouse

Rat

SNORD43, SNORD44, SNORD48

Snord68, Z38 and Z39

Z38, Z39 and U6

For a list of available Probe Sets for normalization small RNAs, please go to www.affymetrix.com/panomics

Guidelines for Data Analysis

Normalizing

Gene Expression

Data

To normalize gene expression data:

Step Action

1

2

Calculate Average Signal - Background (S-B) for samples and normalization RNAs.

Note that background is defined as well containing all assay components except for target miRNA or samples.

Calculate Normalized Signal = S-B of sample/S-B of normalization RNAs.

NOTE:

If multiple normalization RNAs are measured, the geometric mean of backgroundsubtracted AVG housekeeping RNA signals may be used for data normalization.

11

Calculating

Relative Fold

Change of miRNA

Expression

To calculate the relative fold change of target miRNA expression in treated versus untreated samples:

Step Action

1

Run sample of interest using probe sets of normalization small RNA and target miRNA.

2

3

Normalize miRNA expression data as described in

Normalizing Gene Expression Data on page 10 .

Divide the normalized value for the treated sample by the normalized value for the untreated sample.

4

5

Optional. Several normalization small RNA can be run from the same sample and their

Geometric Mean used for normalization purposes.

Optional. Normalization can also be performed for mRNA using the QuantiGene Assay with the same sample prepared for miRNA.

NOTE:

Given that the temperature of the two assays (mRNA and miRNA) will be different, detection of mRNA and miRNA cannot be performed on the same plate, therefore plan on using separate plates for each.

Calculating miRNA

Copy Number/Well

Determination of miRNA copy number can be achieved accurately and easily using the

QuantiGene 2.0 miRNA assay. In practice, spike recovery experiments are used to assure specific detection of miRNA copy number in complex sample matrices and cell lysates. The

QuantiGene 2.0 miRNA assay has an excellent spike recovery of 100 ± 20%. Determining absolute copy number compared to relative fold changes provides the ability to compare inter-day, inter-lab and inter-group experiments. This makes the QuantiGene 2.0 miRNA assay practical for both clinical and biomarker translational research.

An example is provided to demonstrate how to determine copy number of miR-145 in

8,000 HeLa cells

:

Step Action

1

2

Use Affymetrix miR-145 Positive Control at 250 pMol (150 million copies/µl) to create an

8-point standard curve in triplicate.

Dilute miR-145 Positive Control in nuclease-free water plus 10 ng/μl Yeast tRNA

(Invitrogen # 15401-011) with a final volume of 20 μl/assay well

Include one background point of 0 copies miR-145 Positive Control

Prepare the lowest dilution point at 2 times above the LOD of miRNA probe set (refer to product insert). For miR-145, the probe set LOD is 30,000 copies, so that the lowest dilution point would be 60,000 copies.

Run standard curve dilutions and sample lysates.

12 QuantiGene ® 2.0 miRNA Assay User Manual

Step Action

3

Determine (signal – background) for standard curve dilutions and samples. Background is assay wells of 0 copies miR-145.

Standard Curve HeLa Lysate

(8,000 cells) miR-145 (LOD =

30,000 copies)

AVG Signal AVG

(Signal –

Background)

AVG

(Signal –

Background)

4,819,496 0

60,000

120,000

600,000

3,000,000

15,000,000

30,000,000

60,000,000

38,227

83,453

127,236

478,620

2,164,208

9,767,725

19,289,475

39,714,450

45,226

89,009

440,393

2,125,981

9,729,498

19,251,248

39,676,223

4

Plot a graph of the signal-background (y-axis) and miRNA copy number (x-axis).

Standard curve for miR-145 Positive Control:

13

Step Action

5

Using linear regression curve fitting (Microsoft Excel or other program) to determine the linear equation and regression coefficient (R 2 ). Note that the R 2 must be equal or greater than 0.96.

To calculate miR-145 copy number /cell using linear curve fitting equation:

Y = 0.6576X – 25773

X = (Y+25773)/0.6576

If Y value: 4,819,496 (signal – background)

Then X value: 4,819,496 + 25773/0.6576 = 7,720,907 copies/ 8,000 cells

Therefore, copy number/cell = 7,720,907 copies/8,000 cells = 965 copies/cell

% of Spike

Recovery

Spike recovery experiments determine the assay’s ability to capture and detect a specific miRNA target in a complex matrices of lysates consisting of proteins, lipids, carbohydrates, and nucleic acids. The QuantiGene 2.0 miRNA Assay must meet the spike recovery of 100

± 20% or a range of 80 – 120%.

To calculate spike recovery:

Step Action

1

Prepare serial dilutions of miRNA Positive Control (from Affymetrix) in lysates or homogenates for spiked samples. Include 0 miRNA as background.

If preparing spiked samples in lysates, dilute Affymetrix miRNA Positive Control in the lysate of interest with a final volume of 80 μl/assay well.

If preparing spiked samples in homogenates, dilute Affymetrix miRNA Positive Control in the homogenate of interest with a final volume of 40 μl/assay well.

2

3

Prepare serial dilutions of miRNA Positive Control (from Affymetrix) in assay buffer for unspiked samples. Include 0 miRNA as background.

If preparing unspiked samples for lysates, dilute Affymetrix miRNA Positive Control in

Diluted Lysis Mixture plus 2.5 ng/μl of Yeast tRNA (Invitrogen # 15401-011) with a final volume of 80 μl/assay well.

If preparing unspiked samples for homogenates, dilute Affymetrix miRNA Positive

Control in Homogenizing Solution plus 5 ng/μl of Yeast tRNA (Invitrogen # 15401-011) with a final volume of 40 μl/assay well.

Run assay, acquire signal and determine background-subtracted signal for spiked and unspiked miRNA

14 QuantiGene ® 2.0 miRNA Assay User Manual

Step Action

4

To determine % spike recovery:

(spiked S-B/unspiked S-B) x 100 = Must be within 80 – 120%

S-B = signal – background (0 miRNA)

NOTE:

Any endogenous miRNA may interfere with the spike recovery results. We have designed a negative miRNA sequence derived from bacterial gene dapB (Affymetrix P/N SMC-10180) to perform spike recovery in various animal and plant lysates. We have also demonstrated the spike recovery of human miR-93 in Corn Husk lysate. See data below.

Spike recovery for dapB miRNA from Corn Husk lysate:

dapB miRNA in

Spike recovery for dapB miRNA from Stomach Tissue lysate:

dapB miRNA in

Step Action

Spike recovery for miR-93 miRNA from Corn Husk lysate:

15

16 QuantiGene ® 2.0 miRNA Assay User Manual

3

QuantiGene 2.0 miRNA Assay Procedure

Assay Workflow

Table 3.1

Step Tasks

Prepare samples a

1

2

Capture miRNA

Dilute samples

Prepare Working Probes Sets

Dispense Working Probe Sets, samples, and controls into Capture Plate

Hybridize overnight

3

Amplify and detect signal

Wash away unbound material

Sequentially hybridize 2.0 PreAmp, Amp, and Label Probe

Add 2.0 Substrate, incubate, and read signal

a For a procedure refer to Appropriate QuantiGene Sample Processing Kit for preparing cultured cell lysate, blood lysates and tissue homogenates. Follow standard laboratory methods for purification of RNA. Use samples immediately, or store at –80 °C until ready to use.

Capturing Target miRNA

Refer to the appropriate procedure for your sample type:

Cultured cell or blood lysates

Fresh, frozen or FFPE tissue homogenates

Total RNA or purified miRNA

Capturing Target miRNA from

Cultured Cell or

Whole Blood/

PAXgene Blood

:

To capture target miRNA from cultured cell or blood lysates:

Step Action

1

Prepare reagents:

Probe Set(s) and Blocking Reagent. Thaw, vortex briefly to mix, then briefly centrifuge to collect contents at the bottom of the tubes. Keep on ice until use.

Cultured cell or whole blood/PAXgene blood lysate(s). If previously frozen, thaw at room temperature followed by incubation at 37 °C for 15 – 30 minutes. Vortex briefly, then leave at room temperature until use.

Lysis Mixture. Re-dissolve any precipitates by incubating at 37 °C for 30 minutes followed by gentle swirling.

Remove Capture Plate from 4 °C and place on the benchtop to warm completely to room temperature (approximately 30 minutes). Do not remove the plate from the sealed foil pouch.

18 QuantiGene ® 2.0 miRNA Assay User Manual

To capture target miRNA from cultured cell or blood lysates:

Step Action

2

Determine sample input.

Estimate sample input based on this chart and the LOD of miRNA target-specific probe set from the package insert.

Recommended Sample Input

(based on LOD = 3,000 Copies of miRNA Probe Set) miRNA (copies per cell) Cultured Cells

(number of cells)

Whole Blood/PAXgene

Blood Lysate (μL)

< 10

100

> 1,000

6,000

600

60

80 a

80

8

3

a May not have sensitivity required.

If appropriate, based on the expression level of target miRNA of interest, dilute sample with Diluted Lysis Mixture (1 volume of Lysis Mixture plus 2 volumes of nuclease-free water, prepared fresh) so that the final desired amount of sample present in a volume of

80 μl/assay well.

Prepare Working Probe Set:

Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed. Scale according to the number of assays to be run.

NOTE:

Include 3 wells for assay background controls.

Preparation of Working Probe Sets

1 Well (μL)

11.7

48 Wells a

(μL)

786.0

96 Wells a

(μL)

1,573.0

Reagent

Nuclease-free

Water

Lysis Mixture

Blocking Reagent

CE

LE

Total

a Includes 40% overage

6.7

1.0

0.3

0.3

20.0

447

67.0

20.1

20.1

1,340.2

893.0

134.0

40.2

40.2

2,680.4

19

To capture target miRNA from cultured cell or blood lysates:

Step Action

4

Prepare the Capture Plate:

A. After the Capture Plate reaches room temperature (minimum of 30 minutes at room temperature), open the sealed foil pouch and remove the Capture Plate.

B. Vortex Working Probe Set briefly to mix, then dispense into the Capture Plate.

C. For fewer than 48 wells: Using a single channel pipette and a new tip for each transfer, dispense 20 µL of Working Probe Set into each assay well. Avoid introducing bubbles.

D. For 48 wells or more:

Using a single channel pipette, transfer Working Probe Set to a 25-mL divided reagent reservoir.

NOTE:

Do not pour or reagent shortage will occur.

Using a multichannel pipette and new tips for each transfer, dispense 20 μL of

Working Probe Set into each assay well. Avoid introducing bubbles.

IMPORTANT:

Capture Probe oligonucleotides are conjugated to the surface of

Capture Plate wells. Do not scratch Capture Plate wells with pipette tips.

5

Add sample to the Capture plate:

Using a new pipette tip for each transfer, add 80 µL of sample to each well of the

Capture Plate containing Working Probe Set. Avoid introducing bubbles. Do not mix.

IMPORTANT:

Add 80 μL of Diluted Lysis Mixture (1 volume Lysis Mixture plus 2 volumes nuclease-free water) to 3 wells for the assay background controls. Run background wells for each Probe Set used.

6

Bind target miRNA:

A. Place an adhesive Plate Seal squarely on the plate and seal tightly.

IMPORTANT:

Complete and uniform sealing of the overnight hybridization plate is essential. Use a soft rubber roller or the QuantiGene CTC Plate Sealer. Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal.

B. Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well.

C. Immediately place the Capture Plate in a 46 ± 1 °C incubator to begin the overnight (16–20 hour) hybridization.

IMPORTANT:

Temperature must be 46 ± 1 °C for miRNA assay. Verify temperature using a QuantiGene Incubator Temperature Validation Kit.

Capturing

Target miRNA from

Fresh, Frozen or

FFPE Tissue

Homogenates

To capture target miRNA from fresh, frozen or FFPE tissue homogenates:

Step Action

1

Prepare reagents:

Probe Set(s) and Blocking Reagent. Thaw, vortex briefly to mix, then briefly centrifuge to collect contents at the bottom of the tubes. Keep on ice until use.

Tissue homogenates. If previously frozen, thaw at room temperature followed by incubation at 37 °C for 15 – 30 minutes. Vortex briefly, then leave at room temperature until use.

Lysis Mixture. Re-dissolve any precipitates by incubating at 37 °C for 30 minutes

 followed by gentle swirling.

Remove Capture Plate from 4 °C and place on the benchtop to warm completely to room temperature (approximately 30 minutes). Do not remove the plate from the sealed foil pouch.

20 QuantiGene ® 2.0 miRNA Assay User Manual

To capture target miRNA from fresh, frozen or FFPE tissue homogenates:

Step Action

2

Determine sample input.

Estimate sample input based on this chart and the LOD of miRNA target-specific probe set from the package insert.

Recommended Sample Input

(based on LOD = 3,000 Copies of miRNA Probe Set) miRNA (copies per cell) Tissue Homogenate (μL)

< 10

100

> 1,000

40 a

40

4

3

a May not have sensitivity required.

If appropriate, based on the expression level of target miRNA of interest, dilute tissue homogenates with Homogenizing Solution so that the desired amount of sample present in a volume of 40 µl/assay well.

Prepare Working Probe Set:

Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed. Scale according to the number of assays to be run, and include 40% overage.

NOTE:

Include 3 wells for assay background controls.

Reagent

Nuclease-free

Water

Lysis Mixture

Blocking Reagent

CE

LE

Total

a

Includes 40% overage

Preparation of Working Probe Sets

1 Well (μL) 48 Wells a (μL)

25.1

1,682.0

33.3

1.0

0.3

0.3

60.0

2,233.0

67.0

20.1

20.1

4,022.2

96 Wells a

(μL)

3,363.0

4,467.0

134.0

40.2

40.2

8,044.4

21

To capture target miRNA from fresh, frozen or FFPE tissue homogenates:

Step Action

4

Prepare the Capture Plate:

A. After the Capture Plate reaches room temperature (minimum of 30 minutes at room temperature), open the sealed foil pouch and remove the Capture Plate.

B. Vortex Working Probe Set briefly to mix, then dispense into the Capture Plate.

C. For fewer than 48 wells: Using a single channel pipette and a new tip for each transfer, dispense 60 µL of Working Probe Set into each assay well. Avoid introducing bubbles.

D. For 48 wells or more:

Using a single channel pipette, transfer Working Probe Set to a 25-mL divided reagent reservoir.

IMPORTANT:

Do not pour or reagent shortage will occur.

Using a multichannel pipette and new tips for each transfer, dispense 60 μL of

Working Probe Set into each assay well. Avoid introducing bubbles.

IMPORTANT:

Capture Probe oligonucleotides are conjugated to the surface of

Capture Plate wells. Do not scratch Capture Plate wells with pipette tips.

5

Add sample to the Capture plate:

Using a new pipette tip for each transfer, add 40 µL of sample to each well of the

Capture Plate containing Working Probe Set. Avoid introducing bubbles. Do not mix.

IMPORTANT:

Add 40 μL of Homogenizing Solution to 3 wells for the assay background controls. Run background wells for each Probe Set used.

6

Bind target miRNA:

A. Place an adhesive Plate Seal squarely on the plate and seal tightly.

IMPORTANT:

Complete and uniform sealing of the overnight hybridization plate is essential. Use a soft rubber roller or the QuantiGene CTC Plate Sealer. Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal.

B. Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well.

C. Immediately place the Capture Plate in a 46 ± 1 °C incubator to begin the overnight (16–20 hour) hybridization.

IMPORTANT:

Temperature must be 46 ± 1 °C for miRNA assay. Verify temperature using a QuantiGene Incubator Temperature Validation Kit.

Capturing

Target miRNA from Total RNA or

Purified miRNA

:

To capture target miRNA from purified RNA preparations:

Step Action

1

Prepare reagents:

Probe Set(s) and Blocking Reagent. Thaw, vortex briefly to mix, then briefly

 centrifuge to collect contents at the bottom of the tubes. Keep on ice until use.

RNA sample(s). If previously frozen, thaw on ice.

Lysis Mixture. Re-dissolve any precipitates by incubating at 37 °C for 30 minutes followed by gentle swirling.

Remove Capture Plate from 4 °C and place on the benchtop to warm completely to room temperature (approximately 30 minutes). Do not remove the plate from the sealed foil pouch.

22 QuantiGene ® 2.0 miRNA Assay User Manual

To capture target miRNA from purified RNA preparations:

Step Action

2

Determine sample input.

Estimate sample input based on this chart and the LOD of miRNA target-specific probe set from the package insert.

Recommended Sample Input

(based on LOD = 3,000 Copies of miRNA Probe Set) miRNA (copies per cell) Total RNA (ng)

< 10

100

> 1,000

100 a

10

1

3

a 100 ng of RNA is approximately equivalent to 5,000 cells.

Dilute RNA in nuclease-free water so that the desired amount of RNA is present in a volume of 20 µL/assay well based on expression level of target miRNA of interest.

Prepare Working Probe Set:

Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed. Scale according to the number of assays to be run, and include 40% overage.

IMPORTANT:

Include 3 wells for assay background controls.

Reagent

Nuclease-free

Water

Lysis Mixture

Blocking Reagent

CE

LE

Total

a Includes 40% overage

Preparation of Working Probe Sets

1 Well (μL)

45.1

48 Wells a (μL)

3,022.0

33.3

1.0

0.3

0.3

80.0

2,233.0

67.0

20.1

20.1

5362.2

96 Wells a (μL)

6,043.0

4,467.0

134.0

40.2

40.2

10,724.4

23

To capture target miRNA from purified RNA preparations:

Step Action

4

Prepare the Capture Plate:

A. After the Capture Plate reaches room temperature (minimum of 30 minutes at room temperature), open the sealed foil pouch and remove the Capture Plate.

B. Vortex Working Probe Set briefly to mix, then dispense into the Capture Plate.

C. For fewer than 48 wells: Using a single channel pipette and a new tip for each transfer, dispense 80 µL of Working Probe Set into each assay well. Avoid introducing bubbles.

D. For 48 wells or more:

Using a single channel pipette, transfer Working Probe Set to a reagent reservoir.

IMPORTANT:

Do not pour or reagent shortage will occur.

Using a multichannel pipette and new tips for each transfer, dispense 80 μL of

Working Probe Set into each assay well. Avoid introducing bubbles.

IMPORTANT:

Capture Probe oligonucleotides are conjugated to the surface of

Capture Plate wells. Do not scratch Capture Plate wells with pipette tips.

5

Add sample to the Capture plate:

Using a new pipette tip for each transfer, add 20 µL of sample to each well of the

Capture Plate containing Working Probe Set. Avoid introducing bubbles. Do not mix.

IMPORTANT:

Add 20 μL of nuclease-free water to 3 wells for the assay background controls. Run background wells for each Probe Set used.

6

Bind target miRNA:

A. Place an adhesive Plate Seal squarely on the plate and seal tightly.

IMPORTANT:

Complete and uniform sealing of the overnight hybridization plate is essential. Use a soft rubber roller or the QuantiGene CTC Plate Sealer. Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal.

B. Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well.

C. Immediately place the Capture Plate in a 46 ± 1 °C incubator to begin the overnight (16–20 hour) hybridization.

IMPORTANT:

Temperature must be 46 ± 1 °C for miRNA assay. Verify temperature using a QuantiGene Incubator Temperature Validation Kit.

Signal Amplification and Detection

These instructions are for processing a single Capture Plate using multichannel pipettes and reagent reservoirs. To process more than one Capture Plate, scale reagents accordingly. If using a 50-plate kit, scale reagent preparations for a minimum of 10 plates per run, or reagent shortages will occur.

IMPORTANT: Do not let the Capture Plate(s) stand dry for more than 5 minutes at any point in this procedure.

IMPORTANT: Incubation temperatures must be 46 ± 1 °C. Verify temperatures using a

QuantiGene Incubator Temperature Validation Kit.

IMPORTANT: If using a recommended plate washer, centrifugation of the Capture Plate after washing steps is not necessary.

24 QuantiGene ® 2.0 miRNA Assay User Manual

Preparing

Wash Buffer

To prepare the wash buffer:

Step Action

1

Add to a 500-mL graduated cylinder, in this order:

A. 400 mL double-distilled water (ddH

2

O)

B. 1.5 mL Wash Comp 1

C. 2.5 mL Wash Comp 2

D. Bring volume to 500 mL with ddH

2

O.

IMPORTANT:

Scale preparation according to the number of plates to be processed.

500 mL is sufficient for processing one Capture Plate.

2

Transfer to a 500-mL bottle and invert to mix. Do not store unused Wash Buffer. Make

Wash Buffer fresh daily

Hybridizing the

2.0 PreAmplifier

To hybridize the 2.0 PreAmplifier:

Step Action

1

Prepare PreAmp Working Reagent:

2

A. Thaw 2.0 PreAmp, then centrifuge briefly to collect the contents at the bottom of the tube.

B. Add 11 µL of 2.0 PreAmp to 11 mL of Amplifier/Label Probe Diluent.

C. Invert to mix.

D. Keep at room temperature until use.

Wash the Capture Plate:

A. Remove the Capture Plate from the incubator and remove the Plate Seal.

B. Add 200 µL/well of Wash Buffer.

C. Invert the Capture Plate over an appropriate receptacle (for example, a

BioHazard container) and expel the contents forcibly.

D. Firmly tap the inverted plate on a clean paper towel to dry.

E. Repeat steps 2B – 2D two more times using 300 µL/well of Wash Buffer.

IMPORTANT:

For recommendations on automated plate washing, see

Alternative

Plate Washing Method

on page 31

.

3

4

5

Remove all traces of Wash Buffer:

A. Place inverted plate with a dry paper towel into the centrifuge and centrifuge at

240 x g for 1 minute at room temperature. Use maximum acceleration and brake settings available.

IMPORTANT:

Do not exceed 240 x g for 1 minute.

B. Proceed to the next step immediately.

Add 100 µL of 2.0 PreAmp Working Reagent to each well of the Capture Plate.

Seal the Capture Plate with a Plate Seal and incubate at 46 ± 1°C for 60 minutes

25

Hybridizing the

2.0 Amplifier

:

To hybridize the 2.0 Amplifier:

Step Action

1

Prepare 2.0 Amp Working Reagent:

2

3

A. Thaw 2.0 Amp, then centrifuge briefly to collect the contents at the bottom of the tube.

B. Add 11 µL of 2.0 Amp to 11 mL of Amplifier/Label Probe Diluent.

C. Invert to mix.

D. Keep at room temperature until use.

Wash the Capture Plate:

A. Remove the Capture Plate from the incubator and remove the Plate Seal.

B. Add 200 µL/well of Wash Buffer.

C. Invert the Capture Plate over an appropriate receptacle (for example, a

BioHazard container) and expel the contents forcibly.

D. Firmly tap the inverted plate on a clean paper towel to dry.

E. Repeat steps 2B – 2D two more times using 300 µL/well of Wash Buffer.

Remove all traces of Wash Buffer:

A. Place inverted plate with a dry paper towel into the centrifuge and centrifuge at

240 x g for 1 minute at room temperature. Use maximum acceleration and brake settings available.

IMPORTANT:

Do not exceed 240 x g for 1 minute.

4

5

B. Proceed to the next step immediately.

Add 100 µL of 2.0 Amp Working Reagent to each well of the Capture Plate.

Seal the Capture Plate with a Plate Seal and incubate at 46 ± 1 °C for 60 minutes.

Hybridizing the

Label Probe

:

To hybridize the Label Probe:

Step Action

1

Prepare Label Probe Working Reagent:

2

3

A. Centrifuge Label Probe briefly to collect the contents to the bottom of the tube.

B. Add 11 µL of Label Probe to 11 mL of Amplifier/Label Probe Diluent.

C. Invert to mix.

D. Keep at room temperature until use.

Wash the Capture Plate:

A. Remove the Capture Plate from the incubator and remove the Plate Seal.

B. Add 200 µL/well of Wash Buffer.

C. Invert the Capture Plate over an appropriate receptacle (for example, a

BioHazard container) and expel the contents forcibly.

D. Firmly tap the inverted plate on a clean paper towel to dry.

E. Repeat steps 2B – 2D two more times using 300 µL/well of Wash Buffer.

Remove all traces of Wash Buffer:

A. Place inverted plate with a dry paper towel into the centrifuge and centrifuge at

240 x g for 1 minute at room temperature. Use maximum acceleration and brake settings available.

IMPORTANT:

Do not exceed 240 x g for 1 minute.

4

B. Proceed to the next step immediately.

Add 100 µL of Label Probe Working Reagent to each well of the Capture Plate.

26 QuantiGene ® 2.0 miRNA Assay User Manual

To hybridize the Label Probe:

Step Action

5

Seal the Capture Plate with a Plate Seal and incubate at 46 ± 1 °C for 60 minutes.

IMPORTANT:

During this incubation, remove 2.0 Substrate from 4 °C and allow it to warm to room temperature.

Adding the 2.0 Substrate and Detecting

Signal

To add the 2.0 substrate and detecting signal:

Step Action

1

Wash the Capture Plate:

2

A. Remove the Capture Plate from the incubator and remove the Plate Seal.

B. Add 200 µL/well of Wash Buffer.

C. Invert the Capture Plate over an appropriate receptacle

(for example, a BioHazard container) and expel the contents forcibly.

D. Firmly tap the inverted plate on a clean paper towel to dry.

E. Repeat steps 2B – 2D two more times using 300 µL/well of Wash Buffer.

Remove all traces of Wash Buffer:

A. Place inverted plate with a dry paper towel into the centrifuge and centrifuge at

240 x g for 1 minute at room temperature. Use maximum acceleration and brake settings available.

IMPORTANT:

Do not exceed 240 x g for 1 minute.

3

4

5

IMPORTANT:

Ensure that 2.0 Substrate is at room temperature before use and that the luminometer is ready for use.

B. Proceed to the next step immediately.

Add 100 µL of 2.0 Substrate to each well of the Capture Plate.

Seal the Capture Plate with a Plate Seal and incubate at room temperature for

5 minutes.

Remove the Plate Seal, place the Capture Plate in the luminometer, and read. Set integration (read) time to 0.2 seconds. For best results, read plate within 15 minutes.

NOTE:

Depending upon luminometer used, some adjustments in integration time may be required to obtain better signal to background ratio and linearity.

4

Troubleshooting

Low Assay

Signal or Poor

Sensitivity

Table 4.1 Troubleshooting Low Assay Signal or Poor Sensitivity

Probable Cause Recommended Action

Number of target miRNA molecules below limit of detection

Signal amplification reagent incorrectly prepared

Inappropriate hybridization temperature

Inactivation of alkaline phosphatase

Expired reagents were used

Luminometer does not have the required sensitivity

Increase the sample input. Verify complete cell lysis (see

Optimizing Sample Input for QuantiGene miRNA Assay on page 9 ).

Dilute 2.0 PreAmp, 2.0 Amp, and Label Probe in Amplifier/Label

Probe diluent.

Hybridization reactions must be carried out at 46 ± 1 °C as directed in protocol.

Use a QuantiGene Incubator Temperature Validation Kit to verify and monitor the temperature.

Do not exceed 50 °C after the addition of Label Probe. Do not allow the Capture Plate to stand dry for more than 5 minutes once the signal amplification and detection procedure has started.

Reagents are good for up to 6 months from date of receipt.

Only use luminometers that meet or exceed the minimum

performance specifications (see

Optimizing Sample Input for

QuantiGene miRNA Assay on page 9

).

Non-Uniform

Signal Across the Plate

Table 4.2 Troubleshooting Non-Uniform Signal Across the Plate

Probable Cause Recommended Action

Temperature gradients within the incubator

Temperature gradients on

Capture Plate at time of reading

Incomplete sealing during overnight hybridization

Capture Plates exposed to moisture prior to the assay

Variable salt concentrations

Verify that the incubator maintains a constant, even temperature throughout the incubator. Avoid opening and closing the incubator door during hybridization steps.

Read plate at room temperature.

If luminometer has heating capability, ensure that this function is turned off and indicates room temperature.

Use the CTC Plate Sealer for robust plate sealing (Affymetrix P/

N QG0400).

Ensure numbers and letters are clearly visible from under the foil seal.

Verify that the supplied plate seal was used.

Allow the Capture Plate to come to room temperature for 30 minutes before opening the sealed foil pouch to avoid condensation.

Hybridization is affected by salt. When diluting samples, always use the appropriate diluent.

28 QuantiGene ® 2.0 miRNA Assay User Manual

High

Background

Signal

Table 4.3 Troubleshooting High Background Signal

Probable Cause Recommended Action

Residual Wash Buffer Ensure that the plate wash method completely removes all residual

Wash Buffer prior to moving to the next step in the procedure.

Incorrect temperature in the incubator

Expired reagents were used

Verify incubation temperatures using a QuantiGene Incubator

Temperature Validation Kit.

Reagents are good for 6 months from the date of receipt.

Capture Plate sat at room temperature longer than 10 minutes after the addition of sample

Do not let the Capture Plate sit at room temperature for longer than 10 minutes after the addition of the overnight hybridization mixture.

Capture Plate sat at room temperature for longer than

10 minutes before washing

(2nd day)

Cross-talk between wells

Wash the Capture Plate within 10 minutes after removal from the incubator.

Only use Luminometers with cross-talk < 0.001%. Reduce integration time on Luminometer to minimize cross-talk. Move high-expressing samples away from background wells.

Well-To-Well

Variation

Table 4.4 Troubleshooting Assay CVs

Probable Cause Recommended Action

Residual Wash Buffer Ensure that the plate wash method completely removes all residual

Wash Buffer prior to moving to the next step in the procedure.

Scratching of the capture well surface

Minimize contact with the Capture Plate well surfaces during all addition and washing steps.

Cross-talk among neighboring wells during reading

Only use luminometers with cross-talk < 0.001%.

Variable salt concentrations

Inaccurate pipetting

Non-homogenous samples

Samples too viscous to pipet accurately

Hybridization is affected by salt. When diluting samples, always use the appropriate diluent.

Only use calibrated, precision pipettes

Affix tips securely

Use a new tip for each transfer

Pipet slowly and carefully, avoiding bubbles

Warm samples to 37 °C to dissolve any precipitates and vortex briefly before use.

Dilute samples 1 – 2 in the appropriate diluent before use.

29

Day-To-Day

Variation

Table 4.5 Troubleshooting High Inter-Plate CVs

Probable Cause Recommended Action

Variable incubation temperatures

Keep incubation temperatures consistent.

Variable incubation times Keep incubation times consistent, especially for incubation with

2.0 Substrate.

Non-constant time between addition of 2.0 Substrate and plate read

Make sure that time between addition of 2.0 Substrate and plate read is consistent.

30 QuantiGene ® 2.0 miRNA Assay User Manual

A

Alternative Plate Washing Method

Automated Washing Procedure

NOTE: Automated washing of plates might require the purchase of additional Wash Buffer.

Program the BIO-TEK ELx405R washer with settings for the dispense program D3 and the wash programs 44 and 45. Link the dispense program D3 to the wash programs 44 and 45 to yield Link 1 and 2, respectively. Use Link 1 to wash the Capture Plates after the overnight hybridization of the sample with the target-specific Probe Set, after the 2.0 Pre-

Amplifier hybridization and the 2.0 Amplifier hybridization. Use Link 2 to wash the

Capture Plates after the Label Probe hybridization.

Table A.1 ELx405R Washer Settings

Parameter

D3

Program

44 45

Method

Number of cycles

Soak/Shake

Soak duration

Shake before soak

Prime after soak

Prime volume

Prime flow rate

Dispense

Dispense volume

Dispense flow rate

Dispense height

Horizontal dispense position

Horizontal Y dispense position

Bottom wash first

Bottom dispense volume

Bottom flow rate

Bottom dispense height

Bottom dispense position

Prime

Prime volume

Prime flow rate

10

0

No

290

5

115

No

3

Yes

10 seconds

No

No

10

0

No

395

5

115

No

5

Yes

10 seconds

No

No

10

0

No

395

5

115

No

32 QuantiGene ® 2.0 miRNA Assay User Manual

Table A.1 ELx405R Washer Settings

Parameter

Aspiration

Aspirate height

Horizontal aspirate position

Horizontal Y aspirate position

Aspirate rate

Aspirate delay

Crosswise aspirate

Crosswise aspirate on

Crosswise height

Crosswise horizontal position

Final aspirate

Final aspirate delay

D3

Program

44

32

-45

0

5

0

No

Yes

2 seconds

Yes

2 seconds

45

32

-45

0

5

0

No

B

Capture Plate Dimensions

About Capture Plate Dimensions

We provide the Capture Plate dimensions to enable you to setup and validate alternative automated plate washers.

NOTE: The Capture Plate construction adheres to the Society for Biomolecular screening standards.

Figure B.1

B

A

D

C

A

1

E bottom

End

View

Top View

F

E

F

C

D

G

A

B

10.8 mm, Well depth

14.3 mm, Plate height

14.0 mm, Well A1 x-offset

11.2 mm, Well A1 y-offset

9.0 mm, Well-to-well offset

85.5 mm, Plate width

127.8 mm, Plate length

G

34 QuantiGene ® 2.0 miRNA Assay User Manual

A

D

E

B

C

F

G

H

Blank Plate Map

1 2 3 4 5 6 7 8 9 10 11 12

D

E

F

G

H

A

B

C

1 2 3 4 5 6 7 8 9 10 11 12

C

36 QuantiGene ® 2.0 miRNA Assay User Manual

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