Two-Color Microarray-Based Gene Expression Analysis

Two-Color Microarray-Based Gene Expression Analysis
Two-Color
Microarray-Based Gene
Expression Analysis
Low Input Quick Amp
Labeling
Protocol
For use with Agilent Gene Expression oligo microarrays
Version 6.9.1, August 2015
Before you begin, view hands-on
videos of SurePrint procedures at
http://www.agilent.com/genomics/protocolvideos.
Microarrays manufactured with Agilent SurePrint
Technology
For Research Use Only. Not for use in diagnostic procedures.
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© Agilent Technologies, Inc. 2007-2015
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G4140-90050
Edition
Version 6.9.1, August 2015
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Acknowledgements
or send an e-mail to: 
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Safety Notices
CAUTION
A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated conditions are fully understood and met.
WA R N I N G
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly performed or adhered to, could result
in personal injury or death. Do not
proceed beyond a WARNING
notice until the indicated conditions are fully understood and
met.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
In this Guide...
This document describes the Agilent recommended procedures
for the preparation and labeling of complex biological targets
and hybridization, washing, scanning, and feature extraction of
Agilent 60-mer oligonucleotide microarrays for
microarray-based two-color gene expression analysis.
1
Before You Begin
This chapter contains information (such as procedural notes,
safety information, required reagents and equipment) that you
should read and understand before you start an experiment.
2
Procedures
This chapter describes the steps to prepare samples, hybridize,
wash and scan gene expression microarrays, and to extract data
using the Agilent Feature Extraction Software.
3
Supplemental Procedures
This chapter contains instructions for quality assessment of
template RNA and labeled cRNA, and steps to prevent
ozone-related problems.
4
Reference
This chapter contains reference information related to the
protocol.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
3
What’s new in 6.9
• Corrected C Scanner settings.
• Updated QC spec for specific labeling activity.
• Updated product labeling statement.
What’s new in 6.8
• Updated list of supported microarrays and resorted list by
species.
• Expanded instructions to prepare hybridization assembly.
What’s new in 6.7
• Added solvent wash for glassware to prepare for microarray
wash.
• Added list of supported microarrays.
• Added note to calibrate hybridization oven on a regular basis
for accuracy of the collected data.
• Corrected the T7 reagent that is used in labeling reaction
preparation step.
• Updated loading instructions for hybridization oven.
• Added reference to compatibility matrix for non-Agilent
scanners.
What’s new in 6.6
• Support for Agilent SureScan microarray scanner.
4
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Content
1
Before You Begin
7
Procedural Notes 8
Safety Notes
9
Agilent Oligo Microarrays
10
Required Equipment
13
Required Reagents 15
Optional Equipment/Reagents
16
Required Hardware and Software 16
Optional Software 17
2
Procedures
19
Sample Preparation 21
Step 1. Prepare Spike A Mix and Spike B
Step 2. Prepare labeling reaction 27
Step 3. Purify the labeled/amplified RNA
Step 4. Quantify the cRNA 33
23
31
Hybridization 35
Step 1. Prepare the 10× Blocking Agent
35
Step 2. Prepare hybridization samples 36
Step 3. Prepare the hybridization assembly
38
Step 4. Hybridize
40
Microarray Wash 41
Step 1. Add Triton X-102 to Gene Expression wash buffers
Step 2. Prewarm Gene Expression Wash Buffer 2 42
Step 3. Prepare the equipment
42
Step 4. Wash the microarray slides 44
41
Scanning and Feature Extraction
48
Step 1. Scan the slides 48
Step 2. Extract data using Agilent Feature Extraction Software
51
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
5
Contents
3
Supplemental Procedures
57
Absolutely RNA Nanoprep Purification
58
Step 1. Prepare the reagents 58
Step 2. Purify the labeled/amplified RNA
59
Thermocycler Protocol
61
Step 1. Program the thermocycler 61
Step 2. Synthesize cDNA from Total RNA 62
Step 3. Synthesize Fluorescent cRNA Synthesis in vitro
63
Quick Amp Labeling Kit Sample Preparation
64
Step 1. Prepare Spike A Mix and Spike B Mix
66
Step 2. Prepare labeling reaction 69
Step 3. Purify the labeled/amplified RNA 72
Step 4. Quantify the cRNA 72
Quality Assessment of Template RNA and Labeled cRNA
73
Step 1. Prepare for quality assessment
74
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer
75
Step 3. Assess the quality using a NanoDrop Spectrophotometer
78
Preventing Ozone-Related Problems 79
Step 1. Prepare the Stabilization and Drying Solution
Step 2. Wash with Stabilization and Drying Solution
4
Reference
80
81
85
Kit Contents 86
Supplemental User Guides 89
Microarray Handling Tips
90
General Microarray Layout and Orientation 91
Array/Sample tracking microarray slides 94
Related Microarray Reagents 96
6
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Two-Color Microarray-Based Gene Expression Analysis
Protocol
1
Before You Begin
Procedural Notes 8
Safety Notes 9
Agilent Oligo Microarrays 10
Required Equipment 13
Required Reagents 15
Optional Equipment/Reagents 16
Required Hardware and Software 16
Optional Software 17
Make sure you read and understand the information in this chapter and have
the necessary equipment and reagents listed before you start an experiment.
NOTE
Agilent cannot guarantee microarray performance and does not provide technical support
to those who use non-Agilent protocols in processing Agilent microarrays.
7
1

Before You Begin
Procedural Notes
Procedural Notes
• Determine the integrity and purity of the input RNA for labeling and
hybridization prior to use to increase the likelihood of a successful
experiment.
• To prevent contamination of reagents by nucleases, always wear
powder-free laboratory gloves, and use dedicated solutions and pipettors
with nuclease-free aerosol-resistant tips.
• Maintain a clean work area.
• When preparing frozen reagent stock solutions for use:
1 Thaw the aliquot as rapidly as possible without heating above room
temperature, unless otherwise indicated.
2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to
10 seconds to drive the contents off of walls and lid.
3 Store on ice or in a cold block until use, unless otherwise indicated.
• In general, follow Biosafety Level 1 (BL1) safety rules.
8
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Before You Begin
Safety Notes
1
Safety Notes
CAUTION
• Inspect the Stabilization and Drying Solution bottle for chips or cracks prior to use.
Failure to do so may result in bottle breakage.
• Wear appropriate personal protective equipment (PPE) when working in the
laboratory.
WA R N I N G
• Cyanine dye reagents are potential carcinogens. Avoid inhalation, swallowing,
or contact with skin.
• LiCl is toxic and a potential teratogen. May cause harm to breastfed babies.
Possible risk of impaired fertility. Harmful if inhaled, swallowed, or contacts
skin. Target organ: central nervous system. Wear suitable PPE. LiCl is a
component of the 2× Hi-RPM Hybridization Buffer.
• Lithium dodecyl sulfate (LDS) is harmful by inhalation and irritating to eyes,
respiratory system, and skin. Wear suitable PPE. LDS is a component of the 2×
Hi-RPM Hybridization Buffer.
• Triton is harmful if swallowed. Risk of serious damage to eyes. Wear suitable
PPE. Triton is a component of the 2× Hi-RPM Hybridization Buffer and is an
additive in wash buffers.
• Acetonitrile is a flammable liquid and vapor. Harmful if inhaled, swallowed, or
contacts skin. Target organs: liver, kidneys, cardiovascular system, and CNS.
• Stabilization and Drying Solution is toxic and flammable and must be used in a
suitable fume hood. This solution contains acetonitrile and must be disposed of
in a manner consistent with disposal of like solvents. Gloves and eye/face
protection should be used during every step of this protocol, especially when
handling acetonitrile and the Stabilization and Drying Solution.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
9
1

Before You Begin
Agilent Oligo Microarrays
Agilent Oligo Microarrays
For more information on microarray designs visit the following web site:
http://www.chem.agilent.com
To get design files or create a custom design, go to the Agilent eArray web site
at http://earray.chem.agilent.com.
NOTE
Store entire kit at room temperature. After breaking foil on microarray pouch, store
microarray slides at room temperature (in the dark) under a vacuum dessicator or nitrogen
purge box. Do not store microarray slides in open air after breaking foil.
Two, four or eight microarrays printed on each 1-inch × 3-inch glass slide
Catalog SurePrint HD and G3 Microarrays and Microarray Kits
Table 1
10
Catalog SurePrint G3 and HD Microarrays - Human
Part Number
Description
G4851C
G3 Human Gene Expression 8×60K v3 Microarray Kit (3 slides)
G4858A-072363
G3 Human Gene Expression 8×60K v3 Microarray (1 slide)
G4851B
G3 Human Gene Expression 8×60K v2 Microarray Kit (3 slides)
G4858A-039494
G3 Human Gene Expression 8×60K v2 Microarray (1 slide)
G4851A
G3 Human GE 8×60K Microarray Kit (3 slides)
G4858A-028004
G3 Human GE 8×60K Microarray (1 slide)
G4845A
HD Human GE 4×44K v2 Microarray Kit (5 slides)
G2519F-026652
HD Human GE 4×44K v2 Microarray (1 slide)
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Before You Begin
Agilent Oligo Microarrays
Table 2
Catalog SurePrint G3 and HD Microarrays - Mouse
Part Number
Description
G4852B
G3 Mouse GE 8×60K v2 Microarray Kit (3 slides)
G4858A-074809
G3 Mouse GE 8×60K v2 Microarray (1 slide)
G4852A
G3 Mouse GE 8×60K Microarray Kit (3 slides)
G4858A-028005
G3 Mouse GE 8×60K Microarray (1 slide)
G4846A
HD Mouse GE 4×44K v2 Microarray Kit (5-slides)
G2519F-026655
HD Mouse GE 4×44K v2 Microarray (1 slide)
G4122F
HD Whole Mouse Genome Microarray Kit, 4×44K (5 slides)
G2519F-014868
HD Whole Mouse Genome Microarray Kit, 4×44K (1 slide)
G
Table 3
1
Catalog SurePrint G3 and HD Microarrays - Rat
Part Number
Description
G4853B
G3 Rat GE 8×60K v2 Microarray Kit (3 slides)
G4858A-074036
G3 Rat GE 8×60K v2 Microarray (1 slide)
G4853A
G3 Rat GE 8×60K Microarray Kit (3 slides)
G4858A-028279
G3 Rat GE 8×60K Microarray (1 slide)
G4847B
HD Rat GE 4×44K v3 Microarray Kit (3 slides)
G2519F-028282
HD Rat GE 4×44K v3 Microarray (1 slide)
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
11
1
Table 4
12

Before You Begin
Agilent Oligo Microarrays
Catalog SurePrint HD Microarrays and Microarray Kits - Model
Organisms/Non-Human
Part Number
Description
G2519F-021169
Arabidopsis (V4) Gene Expression Microarray, 4×44K (1 slide)
G2519F-021623
Barley Gene Expression Microarray, 4×44K (1 slide)
G2519F-023647
Bovine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022520
Brassica Gene Expression Microarray, 4x44K (1 slide)
G2519F-021193
Canine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-020186
C. elegans (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-026441
Chicken (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022523
Cotton Gene Expression Microarray, 4×44K (1 slide)
G2519F-021791
Drosophila Gene Expression Microarray, 4×44K (1 slide)
G2519F-021322
Horse Gene Expression Microarray, 4×44K (1 slide)
G2519F-015060
Magnaporthe (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022524
Medicago Gene Expression Microarray, 4×44K (1 slide)
G2519F-020449
Mosquito Gene Expression Microarray, 4×44K (1 slide)
G2519F-026440
Porcine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-026806
Rhesus Macaque (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-020908
Rabbit Gene Expression Microarray, 4×44K (1 slide)
G2519F-015241
Rice Gene Expression Microarray, 4×44K (1 slide)
G2519F-020938
Salmon Gene Expression Microarray, 4×44K (1 slide)
G4813A-019921
Sheep Gene Expression Microarray, 8×15K (1 slide)
G2519F-021113
Tobacco Gene Expression Microarray, 4×44K (1 slide)
G2519F-022297
Wheat Gene Expression Microarray, 4×44K (1 slide)
G4813A-016322
Yeast (V2) Gene Expression Microarray, 8×15K (1 slide)
G2519F-026437
Zebrafish (V3) Gene Expression Microarray, 4×44K (1 slide)
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Before You Begin
Required Equipment
1
Custom Microarrays
One, two, four, or eight microarrays printed on each 1-inch × 3-inch glass slide.
Table 5
Custom SurePrint HD Microarrays
Part Number
Description
G4502A
Custom Gene Expression Microarray, 1×244K
G4503A
Custom Gene Expression Microarray, 2×105K
G2514F
Custom Gene Expression Microarray, 4×44K
G2509F
Custom Gene Expression Microarray, 8×15K
Table 6
Custom SurePrint G3 Microarrays
Part Number
Description
G4860A
SurePrint G3 Custom Gene Expression Microarray, 1×1M
G4861A
SurePrint G3 Custom Gene Expression Microarray, 2×400K
G4862A
SurePrint G3 Custom Gene Expression Microarray, 4×180K
G4102A
SurePrint G3 Custom Gene Expression Microarray, 8×60K
Required Equipment
Table 8
Required Equipment
Description
Vendor and part number
Agilent Microarray Scanner
Agilent p/n G4900DA, G2565CA or G2565BA
Hybridization Chamber, stainless
Agilent p/n G2534A
Hybridization gasket slides
1 microarray/slide, 5 slides/box
2 microarrays/slide, 5 slides/box
4 microarrays/slide, 5 slides/box
8 microarrays/slide, 5 slides/box

Agilent p/n G2534-60003
Agilent p/n G2534-60002
Agilent p/n G2534-60011
Agilent p/n G2534-60014
Go to www.agilent.com/genomics to see all
available kit configurations.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
13
1

Before You Begin
Required Equipment
Table 8
Required Equipment (continued)
Description
Vendor and part number
Hybridization oven; temperature set at 65°C
Agilent p/n G2545A
Hybridization oven rotator for Agilent
Microarray Hybridization Chambers
Agilent p/n G2530-60029
nuclease-free 1.5 mL microfuge tube
Ambion p/n 12400 or equivalent
magnetic stir bar (×2)
Corning p/n 401435 or equivalent
magnetic stir plate (×2)
Corning p/n 6795-410 or equivalent
circulating water baths or heat blocks set to
37°C, 40°C, 60°C, 65°C, 70°C, and 80°C,
Corning p/n 6795-420 or equivalent
microcentrifuge
Eppendorf p/n 5417R or equivalent
sterile storage bottle
Nalgene 455-1000 or equivalent
spectrophotometer
NanoDrop p/n ND-1000 UV-VIS or equivalent
micropipettor
Pipetman P-10, P-20, P-200, P-1000 or
equivalent
slide-staining dish, with slide rack (×3)
Thermo Shandon p/n 121 or equivalent
clean forceps
ice bucket
powder-free gloves
sterile, nuclease-free aerosol barrier pipette tips
vortex mixer
timer
nitrogen purge box for slide storage
14
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Before You Begin
Required Reagents
1
Required Reagents
Table 9
Required Reagents
Description
Vendor and part or catalog number
Low Input Quick Amp Labeling Kit, Two-Color
Agilent p/n 5190-2306
RNA Spike-In Kit, Two-Color
Agilent p/n 5188-5279
Gene Expression Hybridization Kit
Agilent p/n 5188-5242
Gene Expression Wash Buffer Kit
Agilent p/n 5188-5327
DNase/RNase-free distilled water
Invitrogen p/n 10977-015
RNeasy Mini Kit
Qiagen p/n 74104
ethanol (95% to 100% molecular biology grade)
Sigma-Aldrich p/n E7023-6×500ML
Milli-Q water or equivalent
isopropyl alcohol (molecular biology grade)
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
15
1

Before You Begin
Optional Equipment/Reagents
Optional Equipment/Reagents
Table 10
Optional Equipment/Reagents
Description
Vendor and part number
2100 Bioanalyzer
Agilent p/n G2939AA
RNA 6000 Nano Assay Kit (RNA Series II Kit)
Agilent p/n 5067-1511
Quick Amp Labeling Kit, Two-Color
Agilent p/n 5190-0444
*
Stabilization and Drying Solution
Agilent p/n 5185-5979
Ozone-Barrier Slide Cover*
Agilent p/n G2505-60550
Absolutely RNA Nanoprep Kit
Agilent p/n 400753
slide box
Corning p/n 07201629
acetonitrile
Sigma p/n 271004-1L
sulfolane
Sigma p/n T22209
thermal cycler
PCR 96-well plate or 0.2 mL PCR tubes
* Recommended when processing microarrays in high ozone environment.
Required Hardware and Software
Table 11
Description
Feature Extraction software 10.7.1 or later
Agilent Scan Control software. Refer to Agilent Scanner user guide for specifications.
For system and supported Internet Explorer/Adobe Reader versions, please see the System
Requirements for your Feature Extraction and Scan Control Software.
16
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Before You Begin
Optional Software
1
Optional Software
Table 12
Description
GeneSpring GX 9.0 or later
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
17
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18
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Two-Color Microarray-Based Gene Expression Analysis
Protocol
2
Procedures
Sample Preparation 21
Step 1. Prepare Spike A Mix and Spike B 23
Step 2. Prepare labeling reaction 27
Step 3. Purify the labeled/amplified RNA 31
Step 4. Quantify the cRNA 33
Hybridization 35
Step 1. Prepare the 10× Blocking Agent 35
Step 2. Prepare hybridization samples 36
Step 3. Prepare the hybridization assembly 38
Microarray Wash 41
Step 1. Add Triton X-102 to Gene Expression wash buffers 41
Step 2. Prewarm Gene Expression Wash Buffer 2 42
Step 3. Prepare the equipment 42
Step 4. Wash the microarray slides 44
Scanning and Feature Extraction 48
Step 1. Scan the slides 48
Step 2. Extract data using Agilent Feature Extraction Software 51
The Agilent Two-Color Microarray-based Gene Expression Analysis uses
cyanine 3- and cyanine 5-labeled targets to measure gene expression in
experimental and control samples. Figure 1 is a standard workflow for sample
preparation and array hybridization design.
19
2

Procedures
Template Total or poly A+ RNA with Spike-In
cDNA synthesis*
cRNA synthesis and amplification*
cRNA purification*
Preparation of hybridization sample
17-hour hybridization (65ºC)
Wash
Scan
Feature Extraction
* Samples can be stored frozen at -80ºC after these steps, if needed.
Figure 1
20
Workflow for sample preparation and array processing.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Sample Preparation
2
Sample Preparation
The Low Input Quick Amp Labeling Kit, Two-Color generates fluorescent
cRNA (complimentary RNA) with a sample input RNA range between 10 ng
and 200 ng of total RNA or a minimum of 5 ng of poly A+ RNA for two-color
processing. The method uses T7 RNA Polymerase Blend (red cap), which
simultaneously amplifies target material and incorporates Cyanine 3-CTP or
Cyanine 5-CTP. Amplification is typically at least a 100-fold from total RNA to
cRNA with the use of this kit.
NOTE
For optimal performance, use pure high quality, intact template total or poly A+ RNA. RNA
that is not pure, as measured by A260/A230 ratio, can lead to poor results and must be
purified. Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on
page 73 for general guidance and procedural recommendations on quality assessment of
template RNA.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
21
2
Procedures
Sample Preparation
Figure 2
22

Schematic of amplified cRNA procedure. Generation of cRNA for a two-color
microarray experiment is shown.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 1. Prepare Spike A Mix and Spike B
2
Step 1. Prepare Spike A Mix and Spike B
(Time required: ~0.5 hours)
Refer to the protocol for RNA Spike-In Kit, Two-Color for in-depth instructions
and troubleshooting advice on how to use two-color spike mixes. This protocol
is available with the RNA Spike-In Kit, Two-Color and can also be downloaded
from the Agilent web site at www.agilent.com/chem/dnamanuals-protocols.
1 Equilibrate water baths to 37°C, 40°C, 65°C, 70°C, and 80°C.
2 Vigorously mix the Spike A Mix and Spike B Mix solutions on a vortex
mixer.
3 Heat at 37°C for 5 minutes, and mix on a vortex mixer once more.
4 Briefly spin in a centrifuge to drive contents to the bottom of the tube prior
to opening. Settlement of the solution on the sides or lid of the tubes may
occur during shipment and storage.
Table 13 provides the dilutions of Spike A Mix and Spike B Mix for four
different starting sample inputs of total RNA or 5 ng of poly A mRNA. Always
label Spike A Mix with cyanine 3 and Spike B Mix with cyanine 5. The dilution
scheme for Spike A Mix with cyanine 3 is the same as the dilution scheme for
Spike B Mix with cyanine 5. The output of the Agilent SpikeIns LogRatio
Statistics table in the QC report, generated when using Agilent Feature
Extraction Software 9.5 or later, uses this orientation. It is built into the
software code and cannot be set by the user. If the polarity is flipped, you will
have to manually adjust the output.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
23
2

Procedures
Step 1. Prepare Spike A Mix and Spike B
Table 13
Dilutions of Spike A Mix for cyanine 3 and Spike B Mix for cyanine 5 labeling
Starting Amount of RNA Serial Dilution
Total
PolyA
RNA (ng) RNA (ng)
First
Second
Third
Fourth
10
1:20
1:40
1:16
1:20
2
25
1:20
1:40
1:16
1:8
2
50
1:20
1:40
1:16
1:4
2
100
1:20
1:40
1:16
1:2
2
200
1:20
1:40
1:16
1:20
1:40
1:16
5
NOTE
Spike Mix Volume to be used
in each labeling reaction (µL)
2
1:2
2
Use RNase-free microfuge tubes and tips. Make sure you dispense at least 2 µL with a
pipette to ensure accuracy.
For example, to prepare the Spike A Mix dilution appropriate for 25 ng of total
RNA starting sample:
1 Create the First Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike A Mix First
Dilution.”
b Mix the thawed Spike A Mix vigorously on a vortex mixer.
c Heat at 37°C in a circulating water bath for 5 minutes.
d Mix the Spike A Mix tube vigorously again on a vortex mixer.
e Spin briefly in a centrifuge to drive contents to the bottom of the tube.
f Into the First Dilution tube, put 2 μL of Spike A Mix stock.
g Add 38 μL of Dilution Buffer provided in the Spike-In kit (1:20).
h Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the First Dilution.
24
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 1. Prepare Spike A Mix and Spike B
2
2 Create the Second Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike A Mix Second
Dilution.”
b Into the Second Dilution tube, put 2 μL of First Dilution.
c Add 78 μL of Dilution Buffer (1:40).
d Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the Second Dilution.
3 Create the Third Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike A Mix Third
Dilution.”
b Into the Third Dilution tube, put 2 μL of Second Dilution.
c Add 30 μL of Dilution Buffer (1:16).
d Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all the liquid at the bottom of the tube. This
tube contains the Third Dilution.
4 Create the Fourth Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike A Mix Fourth
Dilution.”
b Into the Fourth Dilution tube, add 4 μL of Third Dilution to 28 μL of
Dilution Buffer for the Fourth Dilution (1:8).
c Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the Fourth Dilution (now at a 102,400-fold final dilution).
5 Add 2 μL of Fourth Dilution to 25 ng of sample total RNA as listed in
Table 13 and continue with cyanine 3 labeling using the Agilent Low Input
Quick Amp Kit protocol as described in “Step 2. Prepare labeling
reaction” on page 27.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
25
2
Procedures
Step 1. Prepare Spike A Mix and Spike B

Storage of Spike Mix dilutions
Store the RNA Spike-In Kit, Two-Color at –70°C to –80°C in a non-defrosting
freezer for up to 1 year from the date of receipt.
Store the first dilution of the Spike A Mix and Spike B Mix positive controls for
up to 2 months in a non-defrosting freezer at -70°C to -80°C. Do not
freeze/thaw more than eight times. After use, discard the second, third and
fourth dilution tubes.
26
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Prepare labeling reaction
2
Step 2. Prepare labeling reaction
(Time required: ~5.5 hours)
For each assay, make sure that the volume of the total RNA sample plus
diluted RNA spike-in controls does not exceed 3.5 μL. Because the 1× reaction
involves volumes of less than 1 μL, prepare components in a master mix and
divide into the individual assay tubes in volumes >1 μL. When preparing 4
samples, use the 5× master mix. When preparing 8 samples, use the 10×
master mix.
NOTE
The starting input for the Low Input Quick Amp Labeling Kit, Two-Color ranges from 10 ng
to 200 ng of total RNA. For best results, start with at least 25 ng of total RNA for the
4-pack and 8-pack formats, and 50 ng of total RNA for the 1-pack and 2-pack formats. For
the 8-pack microarray format, as little as 10 ng of total RNA can be used to generate high
quality data.
1 Add 10 to 200 ng of total RNA or 5 ng polyA RNA to a 1.5-mL
microcentrifuge tube in a final volume of 1.5 μL. If samples are
concentrated, dilute with water until 10 to 200 ng of total or 5 ng of polyA
RNA is added in a 1.5 μL volume. Dilute the total RNA just prior to use and
store the total RNA at concentrations over 100 ng/μL.
2 Add 2 μL of diluted Spike Mix to each tube. Each tube now contains a total
volume of 3.5 μL.
3 Prepare tubes for both Spike A Mix/Cyanine 3-CTP and Spike B
Mix/Cyanine 5-CTP dyes.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
27
2

Procedures
Step 2. Prepare labeling reaction
4 Prepare T7 Primer Mix and add to sample:
a Mix T7 Primer (green cap) and water as listed in Table 14.
Table 14
T7 Primer Mix
Component
Volume (µL) per
reaction
Volume (µL) per 5
reaction
Volume (µL) per
10 reactions
T7 Primer (green cap)
0.8
4
8
Nuclease-free Water
1
5
10
Total Volume
1.8
9
18
b Add 1.8 μL of T7 Primer Mix into each tube that contain 3.5 μL of total
RNA and diluted RNA spike-in controls. Each tube now contains a total
volume of 5.3 μL.
c Denature the primer and the template by incubating the reaction at 65°C
in a circulating water bath for 10 minutes.
d Put the reactions on ice and incubate for 5 minutes.
5 Prewarm the 5× First Strand Buffer (green cap) at 80°C for 3 to 4 minutes
to ensure adequate resuspensions of the buffer components. For optimal
resuspension, briefly mix on a vortex mixer and spin the tube in a
microcentrifuge to drive down the contents from the tube walls. Keep at
room temperature until needed.
6 Prepare and add cDNA Master Mix:
a Immediately prior to use, add the components in Table 15 to a 1.5-mL
microcentrifuge tube. Use a pipette to gently mix. Keep at room
temperature.
The Affinity Script RNase Block Mix (violet cap) is a blend of enzymes.
Keep the Affinity Script RNase Block Mix (violet cap) on ice and add to
the cDNA Master Mix immediately prior to use.
28
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Prepare labeling reaction
Table 15
2
cDNA Master Mix
Component
Volume (µL)
per reaction
Volume (µL)
per 5 reaction
Volume (µL)
per 10
reactions
5× First Strand Buffer (green cap)
2
10
20
0.1 M DTT (white cap)
1
5
10
10 mM dNTP Mix (green cap)
0.5
2.5
5
Affinity Script RNase Block Mix (violet
cap)
1.2
6
12
Total Volume
4.7
23.5
47
b Briefly spin each sample tube in a microcentrifuge to drive down the
contents from the tube walls and the lid.
c Add 4.7 μL of cDNA Master Mix to each sample tube and mix by pipetting
up and down. Each tube now contains a total volume of 10 μL.
d Incubate samples at 40°C in a circulating water bath for 2 hours.
e Move samples to a 70°C circulating water bath and incubate for
15 minutes.
NOTE
Incubation at 70°C inactivates the AffinityScript enzyme.
f Move samples to ice. Incubate for 5 minutes.
g Spin samples briefly in a microcentrifuge to drive down tube contents
from the tube walls and lid.
Stopping Point
If you do not immediately continue to the next step, store the samples at
-80°C.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
29
2

Procedures
Step 2. Prepare labeling reaction
7 Prepare and add two Transcription Master Mixes, one with Cyanine 3-CTP
and one with Cyanine 5-CTP, in separate 1.5-mL microcentrifuge tubes:
a Immediately prior to use, add the components in Table 16 in the order
listed into a 1.5 mL microcentrifuge tube. Use a pipette to gently mix.
Keep at room temperature.
The T7 RNA Polymerase Blend (red cap) is a blend of enzymes. Keep the
T7 RNA Polymerase Blend (red cap) on ice and add to the Transcription
Master Mixes just before use.
Table 16
Transcription Master Mixes
Component
Volume (µL) per
reaction
Volume (µL) per
5 reaction
Volume (µL)
per 10
reactions
Nuclease-free water (white cap)
0.75
3.75
7.5
5× Transcription Buffer (blue cap)
3.2
16
32
0.1 M DTT (white cap)
0.6
3
6
NTP Mix (blue cap)
1
5
10
T7 RNA Polymerase Blend (red cap)
0.21
1.05
2.1
Cyanine 3-CTP or Cyanine 5-CTP
0.24
1.2
2.4
Total Volume
6
30
60
b Add 6 μL of Transcription Master Mixes with Cyanine 3-CTP to the tube
that contains Spike A Mix. Add 6 μL of Transcription Master Mixes with
Cyanine 5-CTP to the tube that contains Spike B Mix. Gently mix by
pipetting. Each tube now contains a total volume of 16 μL.
c Incubate samples in a circulating water bath at 40°C for 2 hours.
Stopping Point
30
If you do not immediately continue to the next step, store the samples at
-80°C.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 3. Purify the labeled/amplified RNA
2
Step 3. Purify the labeled/amplified RNA
(Time required: ~0.5 hours)
Use the RNeasy Mini Kit to purify the amplified cRNA samples.
If sample concentration causes difficulty, you can use the Absolutely RNA
Nanoprep Kit as an alternative. See “Absolutely RNA Nanoprep
Purification” on page 58.
NOTE
Make sure that ethanol was added to the RPE buffer as specified in the Qiagen manual
before you continue.
1 Add 84 μL of nuclease-free water to your cRNA sample, for a total volume of
100 μL.
2 Add 350 μL of Buffer RLT and mix well by pipetting.
3 Add 250 μL of ethanol (96% to 100% purity) and mix thoroughly by
pipetting. Do not spin in a centrifuge.
4 Transfer the 700 μL of the cRNA sample to an RNeasy Mini Spin Column
(pink) in a Collection Tube (2 ml). Spin the sample in a centrifuge at 4°C for
30 seconds at 13,000 rpm. Discard the flow-through and collection tube.
5 Transfer the RNeasy column to a new Collection Tube (2 ml) and add
500 μL of Buffer RPE (containing ethanol) to the column. Spin the sample
in a centrifuge at 4°C for 30 seconds at 13,000 rpm. Discard the
flow-through. Re-use the collection tube.
6 Add another 500 μL of Buffer RPE to the column. Centrifuge the sample at
4°C for 60 seconds at 13,000 rpm. Discard the flow-through and the
collection tube.
7 If any Buffer RPE remains on or near the frit of the column or on the
outside of the column, transfer the RNeasy column to a new Collection Tube
(1.5 ml) and spin the sample in a centrifuge at 4°C for 30 seconds at
13,000 rpm to remove any remaining traces of Buffer RPE. Discard this
collection tube and use a fresh Collection Tube (1.5 ml) to elute the cleaned
cRNA sample.
CAUTION
Do not discard the final flow-through in the next step. It contains the cRNA sample.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
31
2
Procedures
Step 3. Purify the labeled/amplified RNA

8 Elute the purified cRNA sample by transferring the RNeasy column to a
new Collection Tube (1.5 ml). Add 30 μL RNase-Free Water directly onto the
RNeasy filter membrane. Wait 60 seconds, then centrifuge at 4°C for
30 seconds at 13,000 rpm.
9 Maintain the cRNA sample-containing flow-through on ice. Discard the
RNeasy column.
32
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 4. Quantify the cRNA
2
Step 4. Quantify the cRNA
Use the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 (or
higher) to quantify the cRNA.
1 Start the NanoDrop software.
2 Click the Microarray Measurement tab.
3 Before initializing the instrument as requested by the software, clean the
sample loading area with nuclease-free water.
4 Load 1.0 to 2.0 μL of nuclease-free water to initialize. Then click OK.
5 Once the instrument has initialized, select RNA-40 as the Sample type (use
the drop down menu).
6 Make sure the Recording button is selected. If not, click Recording so that
the readings can be recorded, saved, and printed.
CAUTION
Failure to engage recording causes measurements to be overwritten, with no
possibility of retrieval.
7 Blank the instrument by pipetting 1.0 to 2.0 μL of nuclease-free water (this
can be the same water used to initialize the instrument) and click Blank.
8 Clean the sample loading area with a laboratory wipe. Pipette 1.0 to 2.0 μL
of the sample onto the instrument sample loading area. Type the sample
name in the space provided and click Measure.
Be sure to clean the sample loading area between measurements and
ensure that the baseline is always flat at 0, which is indicated by a thick
black horizontal line. If the baseline deviates from 0 and is no longer a flat
horizontal line, reblank the instrument with nuclease-free water, then
remeasure the sample.
9 Print the results. If printing the results is not possible, record the following
values:
• Cyanine 3 or cyanine 5 dye concentration (pmol/μL)
• RNA absorbance ratio (260 nm/280 nm)
• cRNA concentration (ng/μL)
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
33
2

Procedures
Step 4. Quantify the cRNA
10 Determine the yield and specific activity of each reaction as follows:
a Use the concentration of cRNA (ng/μL) to determine the μg cRNA yield
as follows:
(Concentration of cRNA)  30 μL (elution volume)
----------------------------------------------------------------------------------------------------------------------------------------- = μg of cRNA
1000
b Use the concentrations of cRNA (ng/μL) and cyanine 3 or cyanine 5
(pmol/μL) to determine the specific activity as follows:
Concentration of Cy3 or Cy5
-----------------------------------------------------------------------------  1000 = pmol Cy3 or Cy5 per μg cRNA
Concentration of cRNA
11 Examine the yield and specific activity results. See Table 17 for the
recommended cRNA yields and specific activities for hybridization.
CAUTION
If the specific activity does not meet the requirements listed in Table 17, do not
continue to hybridization. Repeat preparation of cRNA.
Table 17
NOTE
34
Recommended Yields and Specific Activity
Microarray format
Yield (µg)
Specific Activity (pmol Cy3 or Cy5 per µg cRNA)
1-pack
2.5
6
2-pack
1.875
6
4-pack
0.825
6
8-pack
0.825
6
Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for
general guidance and procedural recommendations on quality assessment of labeled
cRNA.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Hybridization
2
Hybridization
An instructional video that shows hybridization and washing steps can be
found at http://genomics.agilent.com. Search for “Running a microarray
experiment”.
If you are a first time user, practice the hybridization process before you begin.
Use water instead of blocking mix, and use a clean microscope slide and a
gasket slide. Make sure you mix and apply the hybridization solution with
minimal bubbles. Practice the hyb assembly and the slide disassembly and
wash.
CAUTION
You must calibrate the hybridization oven regularly for accuracy of the collected data.
Refer to Agilent G2545A Hybridization Calibration Procedure (p/n G2545-90002,
version A1 or higher) for more information.
Step 1. Prepare the 10× Blocking Agent
1 Add 500 μL of nuclease-free water to the vial containing lyophilized 10×
Gene Expression Blocking Agent supplied with the Gene Expression
Hybridization Kit, or add 1250 μL of nuclease-free water to the vial
containing lyophilized large volume 10× Gene Expression Blocking Agent.
2 Gently mix on a vortex mixer. If the pellet does not go into solution
completely, heat the mix for 4 to 5 minutes at 37°C.
3 Drive down any material that sticks to the tube walls or cap by spinning in a
centrifuge for 5 to 10 seconds.
NOTE
Divide the 10× Gene Expression Blocking Agent into aliquots small enough to keep the
freeze-thaw cycle to 5 times or less. Store at -20°C for up to two months. Before use,
repeat step 2 and step 3.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
35
2

Procedures
Step 2. Prepare hybridization samples
Step 2. Prepare hybridization samples
1 Equilibrate water bath to 60°C.
2 For each microarray, add each of the components as indicated in Table 18
or Table 19 to a 1.5 mL nuclease-free microfuge tube:
3 Mix well but gently on a vortex mixer.
NOTE
For 1-pack and 2-pack microarrays, if you did not generate enough labeled cRNA, add the
amount of labeled cRNA to the fragmentation mix such that the same amount is used for
each microarray within the same experiment (at least 1.65 µg).
Table 18
Components
Volume/Mass
1-pack microarrays
Volume/Mass
2-pack microarrays
Cyanine 3-labeled, linearly amplified cRNA
2.5µg
1.875 µg
Cyanine 5-labeled, linearly amplified cRNA
2.5 µg
1.875 µg
10× Gene Expression Blocking Agent
50 µL
25 µL
Nuclease-free water
bring volume to 240 µL
bring volume to 120 µL
25× Fragmentation Buffer
10 µL
5 µL
Total Volume
250 µL
125 µL
Table 19
36
Fragmentation mix for 1-pack or 2-pack microarray formats
Fragmentation mix for 4-pack or 8-pack microarray formats
Components
Volume/Mass
4-pack microarrays
Volume/Mass
8-pack microarrays
Cyanine 3-labeled, linearly amplified cRNA
825 ng
300 ng
Cyanine 5-labeled, linearly amplified cRNA
825 ng
300 ng
10× Gene Expression Blocking Agent
11 µL
5 µL
Nuclease-free water
bring volume to 52.8 µL
bring volume to 24 µL
25× Fragmentation Buffer
2.2 µL
1 µL
Total Volume
55 µL
25 µL
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Prepare hybridization samples
CAUTION
2
Do not incubate sample in the next step for more than 30 minutes. Cooling on ice and
adding the 2× Hi-RPM Hybridization Buffer stops the fragmentation reaction.
4 Incubate at 60°C for exactly 30 minutes to fragment RNA.
5 Immediately cool on ice for one minute.
6 Add 2× Hi-RPM Hybridization Buffer to stop the fragmentation reaction.
See Table 20.
Table 20
Hybridization mix
Volumes per hybridization
Components
1-pack
2-pack
4-pack
8-pack
cRNA from Fragmentation Mix
250 µL
125 µL
55 µL
25 µL
2× Hi-RPM Hybridization Buffer
250 µL
125 µL
55 µL
25 µL
7 Mix well by careful pipetting part way up and down. Do not introduce
bubbles to the mix. The surfactant in the 2× Hi-RPM Hybridization Buffer
easily forms bubbles. Do not mix on a vortex mixer; mixing on a vortex
mixer introduces bubbles.
8 Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge
to drive the sample off the walls and lid and to aid in bubble reduction.
Use immediately. Do not store.
9 Put sample on ice and load onto the array as soon as possible.
Refer to “Microarray Handling Tips” on page 90 for information on how to
safely handle microarrays.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
37
2

Procedures
Step 3. Prepare the hybridization assembly
Step 3. Prepare the hybridization assembly
Refer to the Agilent Microarray Hybridization Chamber User Guide for more
details to load slides, and to assemble and disassemble the chambers. This
user guide is included with the Agilent Microarray Hybridization Chamber Kit
(G2534A) and can also be downloaded from the Agilent web site at
www.genomics.agilent.com. Search for G2534A.
1 Load a clean gasket slide into the Agilent SureHyb chamber base with the
label facing up and aligned with the rectangular section of the chamber
base. Make sure that the gasket slide is flush with the chamber base and is
not ajar.
CAUTION
Do not let the pipette tip or the hybridization solution touch the gasket walls. Allowing
liquid to touch the gasket wall greatly increases the likelihood of gasket leakage.
When you lower the microarray slide on top of the SureHyb gasket slide, make sure
that the two slides are parallel at all times.
2 Slowly dispense the volume of hybridization sample (see Table 21) onto the
gasket well in a “drag and dispense” manner.
• Position the slides so that the barcode label is to your left.
• Load the samples left to right. For 8-pack slides, start with the first row.
The output files will come out in that same order. Refer to “Array/Sample
tracking microarray slides” on page 94 for guidelines on tracking sample
position for multipack slide formats.
• Avoid the introduction of air bubbles to the gasket wells. Air bubbles can
affect the final sample volume and can cause leakage from the gasket
well.
Table 21
Hybridization Sample
Volumes per hybridization
38
Components
1-pack
2-pack
4-pack
8-pack
Volume Prepared
500 µL
250 µL
110 µL
50 µL
Volume to Hybridize
490 µL
240 µL
100 µL
40 µL
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 3. Prepare the hybridization assembly
2
3 If any wells are unused:
a Make a 1× solution of the 2× Hi-RPM Hybridization Buffer.
b Add the volume of 1× Hybridization Buffer equal to the sample volume to
each unused well.
Make sure all wells contain sample or 1× Hybridization Buffer. Empty wells
can cause failure in hybridization.
4 Grip the slide on either end and slowly put the slide “active side” down,
parallel to the SureHyb gasket slide, so that the “Agilent”-labeled barcode is
facing down and the numeric barcode is facing up. Make sure that the
sandwich-pair is properly aligned.
CAUTION
Do not drop the array slide onto the gasket. Doing so increases the chances of
samples mixing between gasket wells.
5 Put the SureHyb chamber cover onto the sandwiched slides and slide the
clamp assembly onto both pieces.
6 Firmly hand-tighten the clamp onto the chamber.
7 Vertically rotate the assembled chamber to wet the gasket and assess the
mobility of the bubbles. If necessary, tap the assembly on a hard surface to
move stationary bubbles.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
39
2
Procedures
Step 4. Hybridize

Step 4. Hybridize
1 Load each assembled chamber into the oven rotator rack. Start from the
center of the rack (position 3 or 4 when counting from the left). Set your
hybridization rotator to rotate at 10 rpm when using 2× Hi-RPM
Hybridization Buffer.
2 Hybridize at 65°C for 17 hours.
40
CAUTION
If you are not loading all the available positions on the hybridization rotator rack, be
sure to balance the loaded hybridization chambers on the rack so that there are an
equal number of empty positions on each of the four rows on the hybridization rack.
NOTE
The Gene Expression Wash Buffer 2 needs to be warmed overnight. Make sure that you
prepare the wash buffer the night before you plan to do the microarray wash. See “Step 2.
Prewarm Gene Expression Wash Buffer 2”.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Microarray Wash
2
Microarray Wash
Step 1. Add Triton X-102 to Gene Expression wash buffers
This step is optional but highly recommended.
The addition of 0.005% Triton X-102 (10%) to the Gene Expression wash
buffers reduces the possibility of array wash artifacts. Add Triton X-102 (10%)
to Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 when
the cubitainer of wash buffer is first opened.
Do this step to both Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2 before use.
1 Open the cardboard box with the cubitainer of wash buffer and carefully
remove the outer and inner caps from the cubitainer.
2 Use a pipette to add 2 mL of the provided Triton X-102 (10%) into the wash
buffer in the cubitainer.
3 Replace the original inner and outer caps and mix the buffer carefully but
thoroughly by inverting the container 5 to 6 times.
4 Carefully remove the outer and inner caps and install the spigot provided
with the wash buffer.
5 Prominently label the wash buffer box to indicate that Triton X-102 (10%)
has been added and indicate the date of addition.
Triton X-102 (10%) can be added to smaller volumes of wash buffer as long as
the final dilution of the 10% Triton X-102 is 0.005% in the Gene Expression
wash buffer solution.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
41
2
Procedures
Step 2. Prewarm Gene Expression Wash Buffer 2

Step 2. Prewarm Gene Expression Wash Buffer 2
Warm the Gene Expression Wash Buffer 2 to 37°C as follows:
1 Dispense 1000 mL of Gene Expression Wash Buffer 2 directly into a sterile
storage bottle. Repeat until you have enough prewarmed Wash Buffer 2
solution for your experiment.
2 Tightly cap the sterile storage bottle and put in a 37°C water bath the night
before washing arrays. Alternatively, remove the plastic cubitainer from the
box and put it in a 37°C water bath the night before washing the arrays.
Step 3. Prepare the equipment
Always use clean equipment when doing the hybridization and wash steps.
Designate and dedicate dishes to two-color experiments.
Solvent wash
Wash staining dishes, racks and stir bars with acetonitrile or isopropyl alcohol
to avoid wash artifacts on your slides and images.
• Use acetonitrile for equipment that was exposed to Stabilization and
Drying Solution.
• Use isopropyl alcohol for equipment that was not exposed to
Stabilization and Drying Solution.
WA R N I N G
Conduct solvent washes in a vented fume hood.
1 Add the slide rack and stir bar to the staining dish.
2 Transfer the staining dish with the slide rack and stir bar to a magnetic stir
plate.
3 Fill the staining dish with 100% acetonitrile or isopropyl alcohol.
4 Turn on the magnetic stir plate and adjust the speed to a setting of 4
(medium speed).
5 Wash for 5 minutes.
6 Discard the solvent as is appropriate for your site.
42
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 3. Prepare the equipment
2
7 Repeat step 1 through step 6.
8 Air dry the staining dish in the vented fume hood.
9 Proceed to “Milli-Q water wash”.
Milli-Q water wash
Wash all dishes, racks, and stir bars with Milli-Q water.
1 Run copious amounts of Milli-Q water through the staining dish.
2 Empty out the water collected in the dish.
3 Repeat step 1 and step 2 at least 5 times, as it is necessary to remove any
traces of contaminating material.
4 Discard the Milli-Q water.
CAUTION
Some detergents may leave fluorescent residue on the dishes. Do not use any
detergent in the washing of the staining dishes. If detergent is used, all traces must
be removed by copiously rinsing with Milli-Q water.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
43
2

Procedures
Step 4. Wash the microarray slides
Step 4. Wash the microarray slides
NOTE
The microarray wash procedure for the Agilent two-color platform must be done in
environments where ozone levels are 5 ppb or less. For Scanner C and Scanner B, if ozone
levels are between 5 to 10 ppb in your laboratory, use the Agilent Ozone Barrier Slide
Cover (described in this topic). If ozone levels exceed 10 ppb, see “Preventing
Ozone-Related Problems” on page 79 for more information.
NOTE
When setting up the apparatus for the washes, be sure to do so near the water bath
containing the pre-warmed Wash 2 solutions.
Table 22 lists the wash conditions for the wash procedure.
Table 22
Wash conditions
Dish
Wash Buffer
Temperature
Disassembly
1
Gene Expression Wash Buffer 1
Room temperature
1st wash
2
Gene Expression Wash Buffer 1
Room temperature
2nd wash
3
Gene Expression Wash Buffer 2
Time
1 minute
*
Elevated temperature
1 minute
* The elevated temperature of the second wash step is usually around 31°C due to cooling by the
room temperature dish and the rack of arrays.
1 Completely fill slide-staining dish #1 with Gene Expression Wash Buffer 1
at room temperature.
2 Put a slide rack into slide-staining dish #2. Add a magnetic stir bar. Fill
slide-staining dish #2 with enough Gene Expression Wash Buffer 1 at room
temperature to cover the slide rack. Put this dish on a magnetic stir plate.
3 Put the empty dish #3 on the stir plate and add a magnetic stir bar. Do not
add the prewarmed (37°C) Gene Expression Wash Buffer 2 until the first
wash step has begun.
4 Remove one hybridization chamber from incubator and record time. Record
whether bubbles formed during hybridization and if all bubbles are rotating
freely.
44
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 4. Wash the microarray slides
2
5 Prepare the hybridization chamber disassembly.
a Put the hybridization chamber assembly on a flat surface and loosen the
thumbscrew, turning counterclockwise.
b Slide off the clamp assembly and remove the chamber cover.
c With gloved fingers, remove the array-gasket sandwich from the chamber
base by grabbing the slides from their ends. Keep the microarray slide
numeric barcode facing up as you quickly transfer the sandwich to
slide-staining dish #1.
d Without letting go of the slides, submerge the array-gasket sandwich into
slide-staining dish #1 containing Gene Expression Wash Buffer 1.
6 With the sandwich completely submerged in
Gene Expression Wash Buffer 1, pry the sandwich open from the barcode
end only:
a Slip one of the blunt ends of the forceps between the slides.
b Gently turn the forceps upwards or downwards to separate the slides.
c Let the gasket slide drop to the bottom of the staining dish.
d Grasp the top corner of the microarray slide, remove the slide, and then
put it into the slide rack in the slide-staining dish #2 that contains
Gene Expression Wash Buffer 1 at room temperature. Transfer the slide
quickly so avoid premature drying of the slides. Touch only the barcode
portion of the microarray slide or its edges!
More effort is needed to separate the 4-pack and 8-pack sandwiched slides
than the 1-pack and 2-pack sandwiched slides.
7 Repeat step 4 through step 6 for up to seven additional slides in the group.
For uniform washing, do up to a maximum of eight disassembly procedures
yielding eight microarray slides.
8 When all slides in the group are placed into the slide rack in slide-staining
dish #2, stir using setting 4 for 1 minute.
9 During this wash step, remove Gene Expression Wash Buffer 2 from the
37°C water bath and pour into the slide-staining dish #3.
10 Transfer slide rack to slide-staining dish #3 that contains
Gene Expression Wash Buffer 2 at elevated temperature. Stir using setting
4, or a moderate speed setting, for 1 minute.
11 Slowly remove the slide rack minimizing droplets on the slides. It should
take 5 to 10 seconds to remove the slide rack. If liquid remains on the
bottom edge of the slide, dab it on a cleaning tissue.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
45
2
Procedures
Step 4. Wash the microarray slides

12 Discard used Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2.
13 Repeat step 1 through step 12 for the next group of eight slides using fresh
Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2
pre-warmed to 37°C.
14 Put the slides in a slide holder.
For SureScan microarray scanner
• Carefully put the end of the slide without the barcode label onto the slide
ledge.
• Gently lower the microarray slide into the slide holder. Make sure that
the active microarray surface (with “Agilent”-labeled barcode) faces up,
toward the slide cover.
• Close the plastic slide cover, pushing on the tab end until you hear it
click.
• For more detailed instruction, refer to the Agilent G4900DA SureScan
Microarray Scanner System User Guide.
Figure 3
Slide in slide holder for SureScan microarray scanner
For Agilent Scanner B or C only:
• In environments in which the ozone level exceeds 5 ppb, immediately put
the slides with active microarray surface (with “Agilent”-labeled
barcode) facing up in a slide holder. Make sure that the slide is not
caught up on any corner. Put an ozone-barrier slide cover on top of the
array as shown in Figure 4. Refer to the Agilent Ozone-Barrier Slide
Cover User Guide (p/n G2505-90550), included with the slide cover, for
more information.
As an alternative, use the Stabilization and Drying Solution. See
“Preventing Ozone-Related Problems” on page 79.
46
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 4. Wash the microarray slides
Figure 4
2
Inserting the ozone-barrier slide cover (shown for Scanner B and Scanner C)
• In environments in which the ozone level is below 5 ppb, put the slides
with Agilent barcode facing up in a slide holder.
15 Scan slides immediately to minimize the impact of environmental oxidants
on signal intensities. If necessary, store slides in orange slide boxes in a
nitrogen purge box, in the dark.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
47
2
Procedures
Scanning and Feature Extraction

Scanning and Feature Extraction
Step 1. Scan the slides
Agilent provides support for Agilent microarrays scanned on select
non-Agilent scanners. Please see “Feature Extraction Compatibility Matrix for
Non Agilent scanners” for scanner compatibility and settings
(http://www.chem.agilent.com/Library/usermanuals/Public/G1662-90043_Sc
annerCompatibilityMatrix.pdf).
Agilent can guarantee the quality of data only if the data comes from Agilent
microarrays scanned on Agilent scanners.
A SureScan or Agilent C microarray scanner is required for SurePrint G3
formats.
To get scanner profiles from Agilent:
• For Scan Control 9.1.3 or later, go to
http://www.genomics.agilent.com/article.jsp?pageId=2610
• For Scan Control 8.x, go to
http://www.genomics.agilent.com/article.jsp?pageId=2074
Agilent SureScan Microarray Scanner
1 Put assembled slide holders into the scanner cassette.
2 Select the appropriate scanner protocol:
• AgilentG3_HiSen_GX_2color (for G3 format, high-sensitivity mode)
• AgilentG3_GX_2color (for G3 format, standard mode)
• AgilentHD_GX_2color (for HD format)
3 Verify that the Scanner status in the main window says Scanner Ready.
4 Click Start Scan.
48
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 1. Scan the slides
2
Agilent C Scanner Settings
1 Put assembled slide holders with or without the ozone-barrier slide cover
into scanner carousel.
2 Select Start Slot m End Slot n where the letter m represents the Start slot
where the first slide is located and the letter n represents the End slot
where the last slide is located.
3 Select Profile AgilentG3_GX_2color (for SurePrint G3 formats) or Profile
AgilentHD_GX_2color (for SurePrint HD formats).
4 Verify scan settings for two-color scans. See Table 23.
CAUTION
Do not scan G3 microarrays with HD format settings. The resolution of the resulting
image will not be high enough for data analysis.
Table 23
C Scanner Scan Settings
For HD Microarray Formats
For G3 Microarray Formats
Dye channel
R+G (red and green)
R+G (red and green)
Scan region
Agilent HD (61 × 21.6 mm)
Agilent HD (61 × 21.6 mm)
Scan resolution
5 µm
3 µm
Tiff file dynamic range
20 bit
20 bit
Red PMT gain
100%
100%
Green PMT gain
100%
100%
5 Verify that Output Path Browse is set for desired location.
6 Verify that the Scanner status in the main window says Scanner Ready.
7 Click Scan Slot m-n on the Scan Control main window where the letter m
represents the Start slot where the first slide is located and the letter n
represents the End slot where the last slide is located.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
49
2

Procedures
Step 1. Scan the slides
Agilent B Scanner Settings
Agilent Scan Control software v7.0.03 is recommended for 5 μm scans of
SurePrint HD formats. The Agilent B Scanner does not support G3
microarrays. For G3 microarrays, use the Agilent C Scanner or SureScan
microarray scanner.
1 Put slide into slide holder, with or without the ozone-barrier slide cover,
with Agilent barcode facing up.
2 Put assembled slide holders into scanner carousel.
3 Verify scan settings for two-color scans. See Table 24.
For version 7.X, to change any settings, click Settings > Modify Default
Settings. A window pops up from which you can change the settings.
Table 24
B Scanner Scan Settings
For All Formats
Scan region
Scan Area (61 × 21.6 mm)
Scan resolution (µm)
5
5µm scanning mode
Single Pass
eXtended Dynamic range
(selected)
Dye channel
Red&Green
Red PMT
XDR Hi 100%
XDR Lo 10%
Green PMT
XDR Hi 100%
XDR Lo 10%
4 Select settings for the automatic file naming.
• Prefix1 is set to Instrument Serial Number.
• Prefix2 is set to Array Barcode.
5 Verify that the Scanner status in the main window says Scanner Ready.
6 Click Scan Slot m-n on the Scan Control main window where the letter m
represents the Start slot where the first slide is located and the letter n
represents the End slot where the last slide is located.
50
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Extract data using Agilent Feature Extraction Software
2
Step 2. Extract data using Agilent Feature Extraction Software
Feature Extraction is the process by which information from probe features is
extracted from microarray scan data, allowing researchers to measure gene
expression in their experiments. To get the most recent Feature Extraction
software for gene expression, go to the Agilent web site at
www.agilent.com/chem/fe.
After generating the microarray scan images, extract .tif images using the
Feature Extraction software.
1 Open the Agilent Feature Extraction (FE) program.
To get the most recent Feature Extraction protocols for gene expression, go
to www.agilent.com/chem/feprotocols.
2 Add the images (.tif) to be extracted to the FE Project.
a Click Add New Extraction Set(s) icon on the toolbar or right-click the
Project Explorer and select Add Extraction...
b Browse to the location of the .tif files, select the .tif file(s) and click
Open. To select multiple files, use the Shift or Ctrl key when selecting.
The FE program automatically assigns a default grid template and protocol
for each extraction set, if the following conditions are met:
• For auto assignment of the grid template, the image must be generated
from a Agilent scanner and have an Agilent barcode.
• For auto assignment of the Two-Color Gene Expression FE protocol, the
default Gene Expression protocol must be specified in the FE Grid
Template properties.
To access the FE Grid Template properties, double-click on the grid
template in the Grid Template Browser.
3 Set FE Project Properties.
a Select the Project Properties tab.
b In the General section, enter your name in the Operator text box.
c In the Input section, verify that at least the following default settings as
shown in Figure 5 are selected.
For outputs that can be imported into Rosetta Resolver, select MAGE and
JPEG.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
51
2
Procedures
Step 2. Extract data using Agilent Feature Extraction Software
Figure 5

Default settings in FE 10.7.
4 Check the Extraction Set Configuration.
a Select the Extraction Set Configuration tab.
b Verify that the correct grid template is assigned to each extraction set in
the Grid Name column. To assign a different grid template to an
extraction set, select one from the pull down menu.
If a grid template is not available to select from the pull down menu, you
must add it to the Grid Template Browser. To add, right-click inside the
Grid Template Browser, select Add. Browse for the design file (.xml) and
click Open to load grid template into the FE database.
To update to the latest grid templates via Online Update, right-click Grid
Template Browser and select Online Update. You can also download the
latest grid templates from Agilent web site at
http://earray.chem.agilent.com. After downloading, you must add the
grid templates to the Grid Template Browser.
After a new grid template is added to the Grid Template Browser,
remember to specify the default protocol for the new grid template if you
want the Feature Extraction program to automatically assign a FE
protocol to an extraction set.
c Verify that the correct protocol is assigned to each extraction set in the
Protocol Name column. To assign a different protocol to an extraction
52
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Extract data using Agilent Feature Extraction Software
2
set, select from the pull down menu. The appropriate protocol begins
with “GE2” for two-color analysis.
The protocols automatically distinguish the formats for processing the
data.
If a protocol is not available to select from the pull down menu, you must
import it to the FE Protocol Browser. To import, right-click FE Protocol
Browser, select Import. Browse for the FE protocol (.xml) and click
Open to load the protocol into the FE database. Visit the Agilent web site
at www.agilent.com/chem/feprotocols to download the latest protocols.
NOTE
These FE Protocols were optimized using data from Agilent catalog arrays, which have
many replicated probes and validated Negative Control probes. If custom arrays without
enough replicated probes are used, or arrays with custom probes designated as Negative
Control probes are used, the default FE Protocols may not be optimal.
NOTE
When the Agilent XDR scanned images are added to Feature Extraction software version
9.1 or later, the High and Low images are automatically combined for data extraction.
NOTE
20-bit single images from the C Scanner are equivalent to 16-bit XDR images from the B
Scanner.
5 Save the FE Project (.fep) by selecting File > Save As and browse for
desired location.
6 Verify that the icons for the image files in the FE Project Window no longer
have a red X through them. A red X through the icon indicates that an
extraction protocol was not selected. If needed, reselect the extraction
protocol for that image file.
7 Select Project > Start Extracting.
8 After the extraction is completed successfully, view the QC report for each
extraction set by double-clicking the QC Report link in the Summary
Report tab. Determine whether the grid has been properly placed by
inspecting Spot Finding at the Four Corners of the Array. See Figure 7 and
Figure 8.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
53
2
Procedures
Step 2. Extract data using Agilent Feature Extraction Software

If a QC Metric Set has been assigned to the FE Project, you can view the results
of the metric evaluation in three ways:
• Project Run Summary - includes a summary sentence.
• QC Report - includes both a summary on the header and a table of metric
values.
• QC Chart - includes a view of the values of each metric compared across all
extractions in FE Project.
Refer to the application note Enhanced Quality Assessment Using Agilent
Feature Extraction QC Metric Sets, Thresholds, and Charting Tools (p/n
5989-5952EN) for more details on quality assessment and troubleshooting
with the Feature Extraction QC Report. This technical note can be
downloaded from the Agilent web site at www.agilent.com. Search for the part
number 5989-5952EN.
Automatic Download from eArray
Feature Extraction version 10.7 or higher can automatically download Grid
Templates, protocols and QC metrics (QCM or QCMT). To set this up, in the
eArray Login Setting dialog box, under Advanced Options, click Use eArray
server during extraction. See Figure 6.
Figure 6
54
eArray Login Setting
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Procedures
Step 2. Extract data using Agilent Feature Extraction Software
2
Page 1 of 3
QC Report - Agilent Technologies : 2 Color Gene Expression
Date
Thursday, March 29, 2012 - 17:59
Image
US23502418_253949410005_S01 [1_2]
Protocol
GE2_107_Sep09 (Read Only)
User Name
annlucas
Grid
039494_D_F_20120312
FE Version
10.7.3.1
Sample(red/green)
BG Method
No Background
Background Detrend
On(FeatNCRange, LoPass)
Multiplicative Detrend
True
Dye Norm
Linear Lowess
Linear DyeNorm Factor
2.55(Red) 5.62(Green)
Additive Error
8(Red)10(Green)
Saturation Value
Spot Finding of the Four Corners of the Array
779065 (r), 777726 (g)
Net Signal Statistics
Agilent SpikeIns:
Red
# Saturated Features
Green
0
0
99% of Sig. Distrib.
80231
53209
50% of Sig. Distrib.
11238
5434
511
266
1% of Sig. Distrib.
Non-Control probes:
Grid Normal
Red
Local
Background
Feature
Red
Non Uniform
Population
Green
Red
Green
3
3
18
0
357
261
1825
183
Spatial Distribution of All Outliers on the Array
384 rows x 164 columns
# Saturated Features
Green
0
0
99% of Sig. Distrib.
41066
12323
50% of Sig. Distrib.
86
44
1% of Sig. Distrib.
22
10
Red and Green Background Corrected Signals (Non-Control
Inliers)
# Features (NonCtrl) with BGSubSignals < 0: 8957 (Red); 8195
(Green)
Figure 7
Example of the first page of a QC Report for 8×60K microarray, generated by
Feature Extraction Software
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
55
2

Procedures
Step 2. Extract data using Agilent Feature Extraction Software
QC Report - Agilent Technologies : 2 Color Gene Expression
Date
Wednesday, November 25, 2009 - 10:17
Image
US23502353_251485044524_S01_H [1_2]
Protocol
GE2_107_Sep09 (Read Only)
User Name
Administrator
Grid
014850_D_F_20090416
FE Version
10.7.1.1
Sample(red/green)
BG Method
No Background
Background Detrend
On(FeatNCRange, LoPass)
Multiplicative Detrend
True
Dye Norm
Linear Lowess
Linear DyeNorm Factor
1.87(Red) 4.07(Green)
Additive Error
6(Red)4(Green)
Saturation Value
Spot Finding of the Four Corners of the Array
642409 (r), 639098 (g)
Net Signal Statistics
Agilent SpikeIns:
Red
# Saturated Features
Green
0
0
99% of Sig. Distrib.
182642
24186
50% of Sig. Distrib.
24753
4055
1211
190
1% of Sig. Distrib.
Non-Control probes:
Grid Normal
Red
Local
Background
Feature
Red
Non Uniform
Population
Green
Red
Green
20
28
0
0
108
105
840
762
Spatial Distribution of All Outliers on the Array
532 rows x 85 columns
# FeatureNonUnif (Red or Green) = 32(0.07%)
# Saturated Features
Green
0
0
99% of Sig. Distrib.
48156
24960
50% of Sig. Distrib.
276
117
49
11
1% of Sig. Distrib.
Red and Green Background Corrected Signals (Non-Control
Inliers)
# Features (NonCtrl) with BGSubSignals < 0: 2837 (Red); 2376
(Green)
# GeneNonUnif (Red or Green) = 25 (0.061 %)
•BG NonUniform
•BG Population
•Red FeaturePopulation •Red Feature NonUniform
•Green FeaturePopulation •Green Feature NonUniform
Figure 8
56
Example of the first page of a QC Report for 4×44K microarray, generated by
Feature Extraction Software
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Two-Color Microarray-Based Gene Expression Analysis
Protocol
3
Supplemental Procedures
Absolutely RNA Nanoprep Purification 58
Thermocycler Protocol 61
Quick Amp Labeling Kit Sample Preparation 64
Quality Assessment of Template RNA and Labeled cRNA
Preventing Ozone-Related Problems 79
73
The procedures in this chapter are optional but recommended.
57
3
Supplemental Procedures
Absolutely RNA Nanoprep Purification

Absolutely RNA Nanoprep Purification
As an alternative to the RNeasy Mini Kit, the Absolutely RNA Nanoprep Kit
can be used to purify the amplified cRNA after “Step 2. Prepare labeling
reaction” on page 27. Use the Absolutely RNA Nanoprep Kit when it is
required or to avoid the need to concentrate purified samples. The Absolutely
RNA Nanoprep Kit uses an elution volume of 20 μL.
Step 1. Prepare the reagents
1 Prepare 80% sulfolane:
a Incubate the 100% sulfolane in a 37°C water bath until liquefied.
100% sulfolane is a solid at room temperature. 80% sulfolane solution is a
liquid at room temperature and can be stored at room temperature for at
least a month.
b Add 1 mL of DNase/RNase-free distilled water to 4 mL of 100% sulfolane
to make 5 mL of 80% sulfolane.
5 mL of 80% sulfolane is enough to process 50 RNA preparations (from up
to 0.1 mL lysate each).
2 Prepare 1× high-salt wash buffer:
a Add 16 mL of 100% ethanol to the bottle of 1.67× High Salt Wash Buffer.
b On the 1.67× High Salt Wash Buffer container, mark the check box for 1×
(Ethanol Added).
c Tighten the cap on the container of 1.67× High Salt Wash Buffer and
store at room temperature.
3 Prepare the 1× low-salt wash buffer:
a Add 68 mL of 100% ethanol to the bottle of 5× Low Salt Wash Buffer.
b On the 5× Low Salt Wash Buffer container, mark the check box for 1×
(Ethanol Added).
c Tighten the cap on the container of 5× Low Salt Wash Buffer and store at
room temperature.
58
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Purify the labeled/amplified RNA
3
Step 2. Purify the labeled/amplified RNA
1 Add 100 μL of the Lysis Buffer to each reaction tube for a total volume of
116 μL.
2 Mix on a vortex mixer, or pipette repeatedly until homogenized.
3 Add an equal volume (116 μL) of 80% sulfolane (room temperature) to the
cell lysate. Mix thoroughly on a vortex mixer for 5 seconds.
You must use equal volumes of 80% sulfolane and cell lysate. Mix on a
vortex mixer until the lysate and sulfolane are thoroughly mixed.
4 Put an RNA-binding nano-spin cup into a 2-ml collection tube.
5 Transfer the 80% sulfolane and cell lysate mixture to the RNA-binding
nano-spin cup and snap the RNA Binding Nano Spin Cup Cap onto the top
of the spin cup.
6 Spin the sample in a microcentrifuge at 12,000 rpm for 60 seconds.
7 Remove and keep the RNA-binding nano-spin cup. Discard the filtrate. Put
the RNA-binding nano-spin cup back into the same 2-ml collection tube.
Up to this point, the RNA has been protected from RNases by the presence
of guanidine thiocyanate.
8 Add 300 μL of 1× High-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the RNA-binding nano-spin cup, and spin the sample in a
microcentrifuge at 12,000 rpm for 60 seconds.
CAUTION
The High-Salt Wash Buffer contains the irritant guanidine thiocyanate.
9 Remove and keep the spin cup. Discard the filtrate. Put the spin cup back
into the same 2-mL collection tube.
10 Add 300 μL of 1× Low-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the spin cup, and spin the sample in a microcentrifuge at 12,000 rpm
for 60 seconds.
11 Repeat step 9 and step 10 for a second low-salt wash.
12 Remove and keep the spin cup. Discard the filtrate. Put the RNA-binding
nano-spin cup back into the same 2-ml collection tube.
13 Add 300 μL of 1× Low-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the RNA-binding nano-spin cup, and spin the sample in a
microcentrifuge at 12,000 rpm for 3 minutes to dry the fiber matrix.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
59
3
Supplemental Procedures
Step 2. Purify the labeled/amplified RNA

14 Transfer the spin cup to a fresh 2-ml collection tube.
15 Add 20 μL of Elution Buffer directly onto the fiber matrix inside the
RNA-binding nano-spin cup. Cap the RNA-binding nano-spin cup and
incubate the sample at room temperature for 2 minutes.
NOTE
The Elution Buffer must be added directly onto the fiber matrix so that the buffer can
permeate the entire fiber matrix.
To increase the RNA yield, warm the Elution Buffer to 60°C.
16 Spin the sample in a microcentrifuge at 12,000 rpm for 5 minutes.
17 If needed, repeat the elution step (step 15 and step 16) to increase the yield
of total RNA.
18 Transfer the eluate in the collection tube to a capped microcentrifuge tube
to store the RNA.
The RNA can be stored at -20°C for up to one month, or at -80°C for
long-term storage.
60
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Thermocycler Protocol
3
Thermocycler Protocol
The procedure in this section is an optional thermocycler protocol for the Low
Input Quick Amp Labeling Kit, Two-Color.
Use a thermocycler to label reactions if you have a limited number of water
baths. The use of a thermocycler can slightly lower the yield of cRNA when
compared to the use of water baths.
Step 1. Program the thermocycler
• Store the following programs into your thermocycler:
• Program 1: 65°C for 10 minutes, 4°C hold
• Program 2: 40°C for 2 hours, 70°C for 15 minutes, 4°C hold
• Program 3: 40°C for 2 hours, 4°C hold
Five minutes at 4°C is enough. Hold at that temperature if the reagents for the
next step are not ready.
NOTE
Use a heated lid for optimal results.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
61
3
Supplemental Procedures
Step 2. Synthesize cDNA from Total RNA

Step 2. Synthesize cDNA from Total RNA
(Time required: ~3 hours)
1 Add 25 to 200 ng of total RNA to a 0.2 mL PCR tube or the well of a 96-well
PCR plate in a volume of 1.5 μL. For optimal performance, use at least 25 ng
of input total RNA.
2 Add 2 μL of the diluted Spike A Mix or Spike B Mix. Please refer to Table 13
on page 24 for detailed instructions on the preparation and use of Spike-in
kits.
3 Prepare the T7 Primer (green cap) Master Mix as described in Table 14 on
page 28.
4 Add 1.8 μL of T7 Promoter Primer Mix to the tube that contains 3.5 μL of
total RNA and diluted RNA spike-in controls. Each tube now contains a
total volume of 5.3 μL.
5 Put the tubes in the thermocycler and run Program 1 to denature the
template and anneal the primer.
6 Keep the reaction tubes in the thermocycler at 4°C, or move to bench top
rack on ice.
7 Immediately prior to use, gently mix the components in Table 15 on page 29
in the order listed by pipetting, and keep at room temperature.
NOTE
Prewarm the 5× First Strand Buffer (green cap) by incubating the vial in an 80°C water bath
for 3 to 4 minutes to ensure adequate resuspension of the buffer components. For optimal
resuspension, mix briefly on a vortex mixer and spin the tube briefly in a microcentrifuge to
drive the contents off the tube walls. Keep at room temperature until use.
NOTE
Keep the Affinity Script RNase Block Mix (violet cap) on ice. Do not add the Affinity Script RNase
Block Mix (violet cap) until just before you start the reactions.
8 To each sample tube, add 4.7 μL of cDNA Master Mix for a total volume of
10 μL. Pipette up and down to mix.
9 Put reaction tubes in thermocycler and run Program 2 to synthesize
double-stranded cDNA.
NOTE
62
Incubation at 70°C inactivates the AffinityScript enzyme.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 3. Synthesize Fluorescent cRNA Synthesis in vitro
3
Step 3. Synthesize Fluorescent cRNA Synthesis in vitro
(Time required: ~2.5 hours)
1 Immediately before use, make Master Mix for each cyanine dye:
a Add the first four components listed in Table 16 on page 30 in the order
shown to 1.5-mL nuclease-free microfuge tubes at room temperature.
b Mix thoroughly on a vortex mixer.
c Add the T7 RNA Polymerase Blend (red cap) and Cyanine 3-CTP or
Cyanine 5-CTP.
d Mix gently, but completely, by pipetting up and down without introducing
bubbles.
NOTE
Do not add the T7 RNA Polymerase Blend (red cap) to Transcription Master Mix until just
before you do the reaction.
2 Keep the reaction tubes from step 9 above in the thermocycler at 4°C, or
move to bench top rack on ice.
3 To each sample tube, add 6 μL of Transcription Master Mix. Gently mix by
pipetting up and down. The final volume of the reaction is now 16 μL.
4 Return the reaction tubes to the thermocycler and run Program 3 (“Step 1.
Program the thermocycler” on page 61) to synthesize labeled cRNA.
5 Purify the labeled cRNA as described on “Step 3. Purify the
labeled/amplified RNA” on page 31.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
63
3
Supplemental Procedures
Quick Amp Labeling Kit Sample Preparation

Quick Amp Labeling Kit Sample Preparation
The Low Input Quick Amp Labeling Kit replaces the Quick Amp Labeling Kit,
Two-Color. If you have studies that are ongoing, continue to use the Quick
Amp Labeling preparation steps described in this section. For new studies, use
the Low Input Quick Amp Labeling Kit as described in Chapter 2,
“Procedures”.
The Quick Amp Labeling Kit, Two-Color generates fluorescent cRNA
(complimentary RNA) with a sample input RNA range between 50ng and 5 μg
of total RNA or a minimum of 10 ng of poly A+ RNA for two-color processing.
The method uses T7 RNA Polymerase Blend (red cap), which simultaneously
amplifies target material and incorporates Cyanine 3-CTP or Cyanine 5-CTP.
There is routinely at least a 100-fold RNA amplification with use of this kit.
NOTE
64
For optimal performance, use high quality, intact template total or poly A+ RNA. Please
refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for general
guidance and procedural recommendations on quality assessment of template RNA.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Quick Amp Labeling Kit Sample Preparation
Figure 9
3
Schematic of amplified cRNA procedure. Generation of cRNA for a two-color
microarray experiment is shown.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
65
3

Supplemental Procedures
Step 1. Prepare Spike A Mix and Spike B Mix
Step 1. Prepare Spike A Mix and Spike B Mix
Refer to the protocol for the RNA Spike-In Kit, Two-Color for in-depth
instructions and troubleshooting advice on how to use the spike mixes. This
protocol is available with the RNA Spike-In Kit, Two-Color and can also be
downloaded from the Agilent web site at
www.agilent.com/chem/dnamanuals-protocols.
1 Equilibrate water baths to 37°C, 65°C, 40°C, and 80°C.
2 Mix the Spike A Mix and Spike B Mix solutions vigorously on a vortex mixer.
3 Heat at 37°C for 5 minutes, and mix on a vortex mixer once more.
4 Briefly spin in a centrifuge to drive contents to the bottom of the tube prior
to opening. Settlement of the solution on the sides or lid of the tubes may
occur during shipment and storage.
Table 25 provides the dilutions of Spike A Mix and Spike B Mix for three
different starting sample ranges of total RNA or 0.2 μg of poly A mRNA.
Always label Spike A Mix with cyanine 3 and Spike B Mix with cyanine 5. The
output of the Agilent SpikeIns LogRatio Statistics table in the QC report,
generated when using Agilent Feature Extraction Software 9.5 or higher uses
this orientation. It is built into the software code and cannot be set by the
user. If the polarity is flipped, you will have to manually adjust the output.
Table 25
Starting amount of RNA
Serial dilution
Total RNA
(ng)
First
Second
Third
50-200
1:20
1:40
1:16
2
201-2000
1:20
1:40
1:4
2
1:20
1:40
0
2
>2000
NOTE
66
Dilutions of Spike A Mix for cyanine 3 and Spike B Mix for cyanine 5 labeling
PolyA
RNA (ng)
200
Spike A Mix or Spike B Mix
volume to be used (µL)
Use RNase-free microfuge tubes and tips. Avoid pipetting volumes less than 2 µL to
ensure accuracy.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 1. Prepare Spike A Mix and Spike B Mix
3
For example, to prepare the Spike A Mix dilution appropriate for 200 ng of
total RNA starting sample:
1 Make the First Dilution:
a Mix the thawed Spike A Mix vigorously on a vortex mixer.
b Heat at 37°C in a circulating water bath for 5 minutes.
c Mix the Spike A Mix tube vigorously again on a vortex mixer.
a Label a new sterile 1.5 mL microcentrifuge tube “Spike A Mix First
Dilution.”
b Into the First Dilution tube, put 2 μL of the concentrated Spike A Mix.
c Add 38 μL of the Dilution Buffer (1:20).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the First Dilution.
2 Make the Second Dilution:
a Label a new sterile 1.5 mL microcentrifuge tube “Spike A Mix Second
Dilution.”
b Into the Second Dilution tube, put 2 μL from the First Dilution tube.
c Add 78 μL of the Dilution Buffer (1:40).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the Second Dilution.
3 Make the Third Dilution:
a Label a new sterile 1.5 mL microcentrifuge tube “Spike A Mix Third
Dilution.”
b Into the Third Dilution tube, put 2 μL from the Second Dilution tube.
c Add 30 μL of the Dilution Buffer (1:16).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the Third Dilution.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
67
3
Supplemental Procedures
Step 1. Prepare Spike A Mix and Spike B Mix

Storage of Spike Mix dilutions
Store the RNA Spike-In Kit, Two-Color at –70°C to –80°C in a non-defrosting
freezer for up to 1 year from the date of receipt.
The first dilution of the Spike A Mix and Spike B Mix positive controls can be
stored up to 2 months in a non-defrosting freezer at –70°C to –80°C and
freeze/thawed up to eight times.
After use, discard the second and third dilution tubes.
68
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Prepare labeling reaction
3
Step 2. Prepare labeling reaction
1 Add 50 to 5000 ng of total or polyA RNA to a 1.5-mL microcentrifuge tube in
an appropriate volume of 8.3 μL or less. Dilute samples so that at least 2 μL
of sample is pipetted into the tube.
2 Prepare tubes for both Spike A Mix/Cyanine 3-CTP and Spike B
Mix/Cyanine 5-CTP dyes.
3 Add 1.2 μL of T7 Promoter Primer (green cap). See Table 26.
Table 26
Template (RNA Spike A or B Mix) and T7 Promoter Primer Mix
RNA volume (µL)
Diluted Spike A
(or B) Mix
amounts (µL)
T7 Promoter
primer (µL)
Total volume (µL)
8.3
2
1.2
11.5
4 Add 2 μL of diluted Spike Mix (A or B).
5 Use nuclease-free water to bring the total reaction volume to 11.5 μL.
6 Denature the primer and the template by incubating the reaction at 65°C in
a circulating water bath for 10 minutes.
7 Put the reactions on ice and incubate for 5 minutes.
8 Immediately prior to use, gently mix the components listed in Table 27 for
the cDNA Master Mix by adding in the order indicated, and keep at room
temperature.
9 Prewarm the 5× First Strand Buffer (green cap) at 80°C for 3 to 4 minutes
to ensure adequate resuspensions of the buffer components. For optimal
resuspension, briefly mix on a vortex mixer and spin the tube in a
microcentrifuge to drive down the contents from the tube walls. Keep at
room temperature until needed.
MMLV-RT (violet cap) and RNase Inhibitor (violet cap) are enzymes, which
need to be kept on ice and are to be added to the cDNA Master Mix just
before starting the reactions.
Be sure to use the 10 mM dNTP mix tube from the kit.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
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3

Supplemental Procedures
Step 2. Prepare labeling reaction
Table 27
cDNA Master Mix
Component
Volume (µL) per
reaction
Volume (µL) per 4.5
reactions
5× First Strand Buffer (green cap)
4
18
0.1 M DTT (white cap)
2
9
10 mM dNTP Mix (green cap)
1
4.5
MMLV-RT (violet cap)
1
4.5
RNase Inhibitor (violet cap)
0.5
2.3
Total Volume
8.5
38.3
10 Briefly spin each sample tube in a microcentrifuge to drive down the
contents from the tube walls and the lid.
11 Add 8.5 μL of cDNA Master Mix to each sample tube and mix by pipetting
up and down.
12 Incubate samples at 40°C in a circulating water bath for 2 hours.
13 Move samples to a 65°C circulating water bath and incubate for 15 minutes.
14 Move samples to ice. Incubate for 5 minutes.
15 Spin samples briefly in a microcentrifuge to drive down tube contents from
the tube walls and lid.
16 Immediately prior to use, gently mix the components listed in Table 28 in
the order indicated for the Transcription Master Mix by pipetting at room
temperature.
17 Prewarm the 50% PEG (clear cap) solution at 40°C for 1 minute. For
optimal resuspension, briefly mix on a vortex mixer and spin the tube in a
microcentrifuge to drive down the contents from the tube walls. Careful
pipetting is required to ensure accurate volume. Keep at room temperature
until needed.
RNase Inhibitor (violet cap), inorganic pyrophosphatase, and T7 RNA
polymerase are enzymes, which need to be kept on ice and should be added
to the Transcription Master Mix just before starting the reactions.
70
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Prepare labeling reaction
Table 28
3
Transcription Master Mix
Component
Volume (µL) per
reaction
Volume (µL) per 4.5
reactions
Nuclease-free water
15.3
68.9
4× Transcription Buffer (clear cap)
20
90
0.1 M DTT (white cap)
6
27
NTP Mix (blue cap)
8
36
PEG (clear cap)
6.4
28.8
RNase Inhibitor (violet cap)
0.5
2.3
Inorganic Pyrophosphatase (red cap)
0.6
2.7
T7 RNA Polymerase Blend (red cap)
0.8
3.6
Cyanine 3-CTP
2.4
10.8
Total Volume
60
270
18 Add 60 μL of Transcription Master Mix to each sample tube. Gently mix by
pipetting.
19 Incubate samples in a circulating water bath at 40°C for 2 hours.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
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3
Supplemental Procedures
Step 3. Purify the labeled/amplified RNA

Step 3. Purify the labeled/amplified RNA
Use the RNeasy Mini Kit to purify the amplified cRNA samples.
If sample concentration causes difficulty, you can use the Absolutely RNA
Nanoprep Kit as an alternative. See “Absolutely RNA Nanoprep
Purification” on page 58.
NOTE
Ensure that ethanol was added to the RPE buffer as specified in the Qiagen manual before
proceeding.
1 Add 20 μL of nuclease-free water to your cRNA sample, for a total volume of
100 μL.
2 Go to “Step 3. Purify the labeled/amplified RNA” on page 31. Start from
step 2 to complete this task.
Step 4. Quantify the cRNA
Use the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 to
quantify the cRNA.
1 Go to “Step 4. Quantify the cRNA” on page 33 and complete step 2 through
step 10
2 Examine the yield and specific activity results.
CAUTION
NOTE
If the yield is <825 ng and the specific activity is <8.0 pmol Cy3 per µg cRNA do not
proceed to the hybridization step. Repeat cRNA preparation.
Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for
general guidance and procedural recommendations on quality assessment of labeled
cRNA.
3 Continue to the hybridization step at “Hybridization” on page 35.
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Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Quality Assessment of Template RNA and Labeled cRNA
3
Quality Assessment of Template RNA and Labeled cRNA
This section gives a general guideline for template RNA and labeled cRNA
quality assessment before proceeding with amplification or hybridization.
Although optional, this step is highly recommended.
Make sure you determine the integrity and purity of the input template RNA,
as well as labeled cRNA, before you label/amplify and hybridize respectively.
Use the NanoDrop UV-VIS Spectrophotometer and the Agilent 2100
bioanalyzer. The RNA 6000 Nano LabChip kit can be used to analyze total
RNA, mRNA, or cRNA with the appropriate assay at the assay specified
concentration. For low concentration samples consider using the RNA 6000
Pico LabChip kit.
For the assessment of total RNA quality, the Agilent 2100 Expert Software
automatically provides a RNA Integrity Number (RIN). RIN provides a
quantitative value for RNA integrity that facilitates the standardization of
quality interpretation. Users should define a minimum threshold RIN number
based on correlative data in order to eliminate experimental bias due to poor
RNA quality. Analysis of single stranded RNA, e.g. mRNA and cRNA, provides
information on size distribution and concentration. It allows relative
quantification of fragments within a size range.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
73
3

Supplemental Procedures
Step 1. Prepare for quality assessment
Step 1. Prepare for quality assessment
• Refer to Table 29 and Table 30 to make sure that you have the appropriate
analyzer, kits, and compatible assays.
Table 29
Analyzer and Kits
Description
Vendor and part number
2100 Bioanalyzer
Agilent p/n G2938C or G2939A
RNA 6000 Nano LabChip Kit
Agilent p/n 5067-1511
RNA 6000 Pico LabChip Kit
Agilent p/n 5067-1513
Spectrophotometer
NanoDrop p/n ND-1000 or equivalent
Table 30
Compatible Assays
Description
Compatible Assay
RNA 6000 Nano LabChip Kit
Eukaryote Total RNA Nano Assay Qualitative
range 5 to 500 ng/µL
RNA 6000 Nano LabChip Kit
mRNA Nano Assay*
Qualitative range 25 to 250 ng/µL
RNA 6000 Pico LabChip Kit
Eukaryote Total RNA Pico Assay Qualitative
range 50 to 5000 pg/µL in water
RNA 6000 Pico LabChip Kit
mRNA Pico Assay*
Qualitative range 250 to 5000 pg/µL in water
* The mRNA assays are suitable for analysis of cRNA as well.
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Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer
3
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer
1 Choose the kit and assay according to your needs. Typically the RNA
Nano 6000 kit and assay will be appropriate.
2 Ensure the 2100 bioanalyzer electrodes have been cleaned as instructed in
the reagent kit guide.
3 Start the Agilent 2100 Expert program (version B.02.06 or higher), turn on
the 2100 bioanalyzer and check communication.
4 Prepare the chip, samples and ladder as instructed in the reagent kit guide.
5 Load the prepared chip into the 2100 bioanalyzer and start the run within
five minutes after preparation.
6 Within the instrument context, choose the appropriate assay from the drop
down list.
7 Start the run. Enter sample names and comments in the Data and Assay
context.
8 Verify the results.
Template RNA results (total RNA)
The resulting electropherogram should have at least two distinct peaks
representing the 18S and 28S ribosomal RNA. Additional bands are the lower
marker, and the potentially 5S RNA. Presence of 5S RNA depends on the
purification method generally showing lower abundance in column purified
total RNA (see Figure 10).
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
75
3

Supplemental Procedures
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer
18S
28S
5S
LM
Figure 10
Analysis of (human) total RNA with the Eukaryote total RNA Nano assay using three different
samples with decreasing integrity: Red, RIN 8.4; Blue, RIN 5.9; Green, RIN 3,6. Characteristic
regions for ribosomal peaks and the lower marker (LM) are displayed.
Labeled cRNA
The resulting electropherogram should have a broad band. The majority of
signal for amplified sample should fall into the size range from 200 to 2000
nucleotides. If there isn't a band in this range, and there are distinct bands
less than 200 nucleotides in length, DO NOT proceed with that sample since it
has likely been degraded and will not provide accurate results. See Figure 11.
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Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer
Figure 11
3
Non-fragmented cRNA products from Agilent's Low RNA Input Linear Amplification Kit PLUS.
Concentration was 120 ng/µL according to NanoDrop, 1 µL was analyzed with the mRNA Nano
assay. Red, Cy5 labeled cRNA; Blue, Cy3 labeled cRNA show the same size distribution. Since Cy5
is excited under the 2100 bioanalyzer assay conditions, the Cy5 label reagents generate a dominant
peak at 120 nt. In comparison to Cy3 labeled cRNA, the Cy5 labeled cRNA yields additional
fluorescence in the profile for the same reason.
For general assistance on evaluation of total RNA with emphasis on the RNA
integrity number, see the corresponding application note: “RNA integrity
number (RIN) - Standardization of RNA quality control”, 5989-1165EN.
Additional information on mRNA can be found in the corresponding
application notes: Interpreting mRNA electropherograms, publication
5988-3001EN, and Optimizing cRNA fragmentation for microarray
experiments using the Agilent 2100 bioanalyzer, publication 5988-3119EN.
To download application notes regarding the 2100 bioanalyzer visit Agilent
web site at www.agilent.com/chem/labonachip.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
77
3
Supplemental Procedures
Step 3. Assess the quality using a NanoDrop Spectrophotometer

Step 3. Assess the quality using a NanoDrop Spectrophotometer
Accurate assessment of total RNA quantity and quality are crucial to the
success of an Agilent Gene Expression experiment. High quality RNA should
be free of contaminants such as carbohydrates, proteins, and traces of organic
solvents, and should also be intact with minimal degradation.
Use the NanoDrop ND-1000 UV-VIS Spectrophotometer (or equivalent) to
assess RNA concentration and purity.
UV-VIS Spectrophotometry
1 In the Nanodrop program menu, select Nucleic Acid Measurement, then
select Sample Type to be RNA-40.
2 Use 1.5 μL of nuclease-free water to blank the instrument.
3 Use 1.5 μL of each total RNA sample to measure RNA concentration. Record
the RNA concentration (ng/μL) for each sample.
4 Record the A260/A280 and A260/A230 ratios.
High-quality total RNA samples have an A260/A280 ratio of 1.8 to 2.0, which
indicates the absence of contaminating proteins. They also have an
A260/A230 ratio of >2.0, which indicates the absence of other organic
compounds, such as guanidinium isothiocyanate, alcohol and phenol as
well as cellular contaminants such as carbohydrates.
An A260/A230 ratio of <2.0 can indicate the presence of these contaminants,
which can interfere with the labeling reaction or can lead to inaccurate
quantification of your total RNA.
78
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Preventing Ozone-Related Problems
3
Preventing Ozone-Related Problems
The Agilent two-color platform is robust in environments where the ozone
level is 5 ppb (approximately 10 μg/m3) or less. Beyond this level, ozone can
significantly affect Cy5 signal and compromise microarray performance.
For Scanner C and Scanner B, the Agilent Ozone-Barrier Slide cover is
designed to protect against ozone-induced degradation of cyanine dyes and is
recommended when using Agilent oligo-based microarrays in high-ozone
environments. See step 14 on page 46.
For the Agilent SureScan scanner, two built-in mechanisms minimize dye
signal degradation by ozone and other dye oxidants:
• SureScan slide holder with an integrated ozone barrier in its lid.
• Catalytic ozone decomposition filtering system inside the scanner.
In addition to the ozone barriers, the Agilent Stabilization and Drying
Solution, which is an organic solvent based wash, can reduce background
variability produced by wash artifacts.
The use of the Agilent Stabilization and Drying Solution is described in this
section. Before you begin, make sure that you follow the correct wash
procedure:
Table 31
Wash procedure to follow
Ozone level in your lab
Wash Procedure
Ozone-Barrier Slide
Cover
< 5 ppb
Wash Procedure without Stabilization and Drying Solution.
“Step 4. Wash the microarray slides” on page 44
No
> 5 ppb and < 10 ppb
Wash Procedure without Stabilization and Drying Solution.
“Step 4. Wash the microarray slides” on page 44
Yes
> 10 ppb
Wash Procedure with Stabilization and Drying Solution. 
See “Step 1. Prepare the Stabilization and Drying Solution” on page 80
and “Step 2. Wash with Stabilization and Drying Solution” on page 81.
Yes
For more information, visit www.agilent.com/chem/dnatechnicalnotes to
download the technical note on Improving Microarray Results by Preventing
Ozone-Mediated Fluorescent Signal Degradation (p/n 5989-0875EN).
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
79
3
Supplemental Procedures
Step 1. Prepare the Stabilization and Drying Solution

Step 1. Prepare the Stabilization and Drying Solution
The Agilent Stabilization and Drying Solution contains an ozone scavenging
compound dissolved in acetonitrile. The compound in solution is present in
saturating amounts and may precipitate from the solution under normal
storage conditions. If the solution shows visible precipitation, warming of the
solution will be necessary to redissolve the compound. Washing slides using
Stabilization and Drying Solution showing visible precipitation will have a
profound adverse effect on microarray performance.
WA R N I N G
The Agilent Stabilization and Drying Solution is a flammable liquid. Warming the
solution will increase the generation of ignitable vapors.
Do not use an open flame or a microwave. Do not increase temperature rapidly.
Warm and mix the material away from ignition sources.
Use gloves and eye/face protection in every step of the warming procedures.
WA R N I N G
Failure to follow the outlined process will increase the potential for fire, explosion,
and possible personal injury. Agilent assumes no liability or responsibility for
damage or injury caused by individuals performing this process.
1 Warm the solution slowly in a water bath or a vented conventional oven at
40°C in a closed container with sufficient head space to allow for
expansion.
NOTE
The original container can be used to warm the solution. Container volume is 700 mL and
contains 500 mL of liquid. If a different container is used, maintain or exceed this
headspace/liquid ratio. The time needed to completely redissolve the precipitate is
dependent on the amount of precipitate present, and may require overnight warming if
precipitation is heavy. DO NOT FILTER the Stabilization and Drying solution.
2 If needed, gently mix to obtain a homogeneous solution.
Mix under a vented fume hood away from open flames, or other sources of
ignition. Warm the solution only in a controlled and contained area that
meets local fire code requirements.
3 After the precipitate is completely dissolved, let the covered solution stand
at room temperature, allowing it to equilibrate to room temperature and
make sure that precipitation does not occur prior to use.
80
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Supplemental Procedures
Step 2. Wash with Stabilization and Drying Solution
3
Step 2. Wash with Stabilization and Drying Solution
NOTE
Use fresh Gene Expression Wash Buffer for each wash group (up to eight slides). The
acetonitrile and Stabilization and Drying Solution can be reused for washing of up to three
groups of slides (for a total of 24 slides).
WA R N I N G
The Stabilization and Drying Solution must be set-up in a fume hood. Wash 1 and
Wash 2 set-up areas should be put close to, or preferably in, the same fume hood.
Use gloves and eye/face protection in every step of the warming procedures.
Table 32 lists the wash conditions for the wash procedure with Stabilization
and Drying Solution.
Table 32
Wash conditions
Dish Wash Buffer
Temperature
Disassembly
1
Gene Expression Wash Buffer 1
Room temperature
1st wash
2
Gene Expression Wash Buffer 1
Room temperature
1 minute
2nd wash
3
Gene Expression Wash Buffer 2
Elevated
temperature*
1 minute
Acetonitrile Wash 4
acetonitrile
Room temperature
10 seconds
3rd wash
Stabilization and Drying Solution Room temperature
30 seconds
5
Time
* The elevated temperature of the second wash step is usually around 31°C due to cooling by the
room temperature dish and the rack of arrays.
1 Completely fill slide-staining dish #1 with Gene Expression Wash Buffer 1
at room temperature.
2 Put a slide rack into slide-staining dish #2. Add a magnetic stir bar. Fill
slide-staining dish #2 with enough Gene Expression Wash Buffer 1 at room
temperature to cover the slide rack. Put this dish on a magnetic stir plate.
3 Put the empty dish #3 on the stir plate and add a magnetic stir bar. Do not
add the pre-warmed (37°C) Gene Expression Wash Buffer 2 until the first
wash step has begun.
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
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3
Supplemental Procedures
Step 2. Wash with Stabilization and Drying Solution

4 Fill slide-staining dish #4 approximately three-fourths full with
acetonitrile. Add a magnetic stir bar and put this dish on a magnetic stir
plate.
5 Fill slide-staining dish #5 approximately three-fourths full with
Stabilization and Drying Solution. Add a magnetic stir bar and put this dish
on a magnetic stir plate.
6 Remove one hybridization chamber from incubator and record time. Record
whether bubbles formed during hybridization, and if all bubbles are
rotating freely.
7 Prepare the hybridization chamber disassembly.
a Put the hybridization chamber assembly on a flat surface and loosen the
thumbscrew, turning counter-clockwise.
b Slide off the clamp assembly and remove the chamber cover.
c With gloved fingers, remove the array-gasket sandwich from the chamber
base by grabbing the slides from their ends. Keep the microarray slide
numeric barcode facing up as you quickly transfer the sandwich to
slide-staining dish #1.
d Without letting go of the slides, submerge the array-gasket sandwich into
slide-staining dish #1 containing Gene Expression Wash Buffer 1.
8 With the sandwich completely submerged in
Gene Expression Wash Buffer 1, pry the sandwich open from the barcode
end only:
a Slip one of the blunt ends of the forceps between the slides.
b Gently turn the forceps upwards or downwards to separate the slides.
c Let the gasket slide drop to the bottom of the staining dish.
d Remove the microarray slide and put into slide rack in the slide-staining
dish #2 containing Gene Expression Wash Buffer 1 at room temperature.
Minimize exposure of the slide to air. Touch only the barcode portion of
the microarray slide or its edges!
9 Repeat step 6 through step 8 for up to seven additional slides in the group.
A maximum of eight disassembly procedures yielding eight microarray
slides is advised at one time in order to facilitate uniform washing.
10 When all slides in the group are put into the slide rack in slide-staining dish
#2, stir using setting 4 for 1 minute.
11 During this wash step, remove Gene Expression Wash Buffer 2 from the
37°C water bath and pour into the Wash 2 dish.
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Supplemental Procedures
Step 2. Wash with Stabilization and Drying Solution
3
12 Transfer slide rack to slide-staining dish #3 containing
Gene Expression Wash Buffer 2 at elevated temperature. Stir using setting
4 for 1 minute.
13 Remove the slide rack from Gene Expression Wash Buffer 2 and tilt the
rack slightly to minimize wash buffer carry-over. Immediately transfer the
slide rack to slide-staining dish #4 containing acetonitrile and stir using
setting 4 for less than 10 seconds.
14 Transfer the slide rack to dish #5 filled with Stabilization and Drying
Solution and stir using setting 4 for 30 seconds.
15 Slowly remove the slide rack trying to minimize droplets on the slides. It
should take 5 to 10 seconds to remove the slide rack.
16 Discard used Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2.
17 Repeat steps 1 through 16 for the next group of eight slides using fresh
Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2
pre-warmed to 37°C.
18 Scan slides immediately to minimize the impact of environmental oxidants
on signal intensities. If necessary, store slides in orange slide boxes in a
nitrogen purge box, in the dark.
CAUTION
Dispose of acetonitrile and Stabilization and Drying Solution as flammable solvents.
19 Immediately continue at step 14 on page 46.
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84
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Two-Color Microarray-Based Gene Expression Analysis
Protocol
4
Reference
Kit Contents 86
Supplemental User Guides 89
Microarray Handling Tips 90
General Microarray Layout and Orientation 91
Array/Sample tracking microarray slides 94
Related Microarray Reagents 96
This chapter contains reference information related to the protocol and
Feature Extraction default parameter settings
85
4

Reference
Kit Contents
Kit Contents
The content of the kits used in this protocol (required and optional) are listed
here.
Table 33
Low Input Quick Amp Labeling Kit, Two-Color
Content
T7 Primer (green cap)
5× First Strand Buffer (green cap)
0.1 M DTT (white cap)
10 mM dNTP Mix (green cap)
Affinity Script RNase Block Mix (violet cap)
5× Transcription Buffer (blue cap)
NTP Mix (blue cap)
T7 RNA Polymerase Blend (red cap)
Nuclease-free Water
Cyanine 3-CTP
Cyanine 5-CTP
Table 34
RNA Spike-In Kit, Two-Color
Content
Spike A Mix
Spike B Mix
Dilution Buffer
86
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Reference
Kit Contents
Table 35
4
Gene Expression Hybridization Kit
Content
10× Gene Expression Blocking Agent
25× Fragmentation Buffer
2× Hi-RPM Hybridization Buffer
Table 36
Gene Expression Wash Buffer Kit
Content
Gene Expression Wash Buffer 1
Gene Expression Wash Buffer 2
Triton X-102 (10%)
Table 37
RNeasy Mini Kit
Content
RNeasy Mini Spin Column (pink)
Collection Tube (1.5 ml)
Collection Tube (2 ml)
Buffer RLT
Buffer RW1
Buffer RPE
RNase-Free Water
Table 38
Absolutely RNA Nanoprep Kit
Content
Lysis Buffer
1.67× High Salt Wash Buffer
5× Low Salt Wash Buffer
Elution Buffer
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
87
4

Reference
Kit Contents
Table 38
Absolutely RNA Nanoprep Kit
Content
DNase Reconstitution Buffer (green cap)
DNase Digestion Buffer (green cap)
Beta-Mercaptoethanol (yellow cap)
RNase-free DNase I
RNA-binding nano-spin cup
2-ml collection tube
RNA Binding Nano Spin Cup Cap
Table 39
Quick Amp Labeling Kit, Two-Color
Content
T7 Promoter Primer (green cap)
5× First Strand Buffer (green cap)
0.1 M DTT (white cap)
10 mM dNTP Mix (green cap)
RNase Inhibitor (violet cap)
MMLV-RT (violet cap)
4× Transcription Buffer (clear cap)
NTP Mix (blue cap)
Inorganic Pyrophosphatase (red cap)
T7 RNA Polymerase Blend (red cap)
PEG (clear cap)
Cyanine 3-CTP
Cyanine 5-CTP
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Supplemental User Guides
4
Supplemental User Guides
First-time users of the Agilent oligo microarray system, please refer to the
following user manuals for detailed descriptions and operation
recommendations for each of the hardware and software components used in
the two-color platform workflow. The user guides can be downloaded from the
Agilent web site at www.agilent.com/chem/dnamanuals-protocols.
• Agilent Microarray Hybridization Chamber User Guide
• Hybridization Oven User Manual
• Microarray Scanner System User Guide
• G4900DA SureScan Microarray Scanner User Guide
• Feature Extraction Software Quick Start Guide
• Feature Extraction Software User Guide
• Feature Extraction Software Reference Guide
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Microarray Handling Tips

Microarray Handling Tips
Each microarray is printed on the side of the glass slide containing the
“Agilent”-labeled barcode. This side is called the “active” side. The numeric
barcode is on the inactive side of the slide.
CAUTION
You must familiarize yourself with the assembly and disassembly instructions for use
with the Agilent Microarray Hybridization Chamber (G2534A) and gasket slides.
Practice slide kits are available.
In this “processing and hybridization” procedure, the hybridization mixture is
applied directly to the gasket slide, and not to the active side of the oligo
microarray. Instead, the active side of the oligo microarray is put on top of the
gasket slide to form a “sandwich slide” pair.
To avoid damaging the microarray, always handle glass slides carefully by
their edges. Wear powder-free gloves. Never touch the surfaces of the slides. If
you do, you may cause irreparable damage to the microarray.
Never allow the microarray surface to dry out during the hybridization
process and washing steps.
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General Microarray Layout and Orientation
4
General Microarray Layout and Orientation
Agilent oligo microarray (1 microarray/slide format) as imaged on the Agilent
microarray scanner
Microarrays are printed on the side of the glass labeled with the “Agilent” bar code
(also referenced as "active side" or "front side").
00116789
Agilent Microarray
Scanner scans
through the glass.
(Back side scanning.)
Figure 12
Agilent microarray slide holder for Scanner B
and C (left) or SureScan microarray scanner
(right)
Agilent microarray slide and slide holder. The opposite or “non-active”
numerically barcoded side is shown.
Agilent oligo microarray formats and the resulting “microarray design files”
are based on how the Agilent microarray scanner images 1-inch × 3-inch glass
slides. Agilent designed its microarray scanner to scan through the glass slide
(back side scanning). The glass slide is securely placed in an Agilent
microarray slide holder with the “Agilent” labeled barcode facing the opening
of the slide holder (on SureScan Microarray Scanner G4900DA) or facing the
inside of the slide holder (C scanner G2565CA). In this orientation, the “active
side” containing the microarrays is protected from potential damage by
fingerprints and other elements. Once securely placed, the numeric barcode,
non-active side of the slide, is visible from the outside of the slide holder.
Figure 12 depicts how the Agilent microarray scanner reads the microarrays
and how this relates to the “microarray design files” that Agilent generates
during the manufacturing process of its in situ-synthesized oligonucleotide
microarrays. Thus, if you have a scanner that reads microarrays from the
“front side” of the glass slide, the collection of microarray data points will be
different in relation to the “microarray design files” supplied with the Agilent
oligo microarray kit you purchased. Therefore, please take a moment to
become familiar with the microarray layouts for each of the Agilent oligo
microarrays and the layout information as it pertains to scanning using a
“front side” scanner.
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General Microarray Layout and Orientation

Non-Agilent front side microarray scanners
When imaging Agilent oligo microarray slides, you must determine:
• If the scanner images microarrays by reading them on the “front side” of the
glass slide (“Agilent”-labeled barcode side of the slide) and
• If the image produced by the non-Agilent scanner is oriented in a “portrait”
or “landscape” mode, “Agilent”-labeled barcode left-side, right-side, up or
down, as viewed as an image in the imaging software (see Figure 13).
This changes the feature numbering and location as it relates to the
“microarray design files” found on the CD in each Agilent oligo microarray kit.
Microarray layout maps are available from Agilent. For more information, go
to www.agilent.com/chem/dnamanuals-protocols and download Agilent
Microarray Formats Technical Drawings with Tolerance (publication
G4502-90001). This document contains visual references and guides that will
help you determine the feature numbering as it pertains to your particular
scanner configuration.
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General Microarray Layout and Orientation
4
Front side
bar code up
(portrait)
Agilent
Agilent
Agilent
Front side
bar code left
(landscape)
Front side
bar code right
(landscape)
Agilent
Front side
bar code down
(portrait)
Figure 13
Microarray slide orientation
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Reference
Array/Sample tracking microarray slides
Array/Sample tracking microarray slides
Use the forms below to make notes to track your samples on microarray slides.
Position the gasket slide in the SureHyb chamber base with the label to the
left. Load the samples: top row, left to right, then lower row, left to right. The
array suffix assignments from Feature Extraction will be in the order shown.
L
Arrays
Array 1_1
B
A
R
C
O
D
E
Sample:
Array 1_2
Sample:
Array 1_3
Array 1_4
Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:
Barcode Number __________________________________________________________
Figure 14
94
4-pack microarray slides
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Reference
Array/Sample tracking microarray slides
4
Arrays
Array 1_1
B
A
R
C
O
D
E
Array 1_2
Array 1_3
Array 1_4
p
Sample:
p
Sample:
p
Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:
Sample:
Sample:
Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:
A
Array
2 1
2_1
A
Array 2
2_2
2
A
Array
2_3
2 3
A
Array 2
2_4
4
Barcode Number __________________________________________________________
Figure 15
8-pack microarray slide
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Reference
Related Microarray Reagents
Related Microarray Reagents
96
Description
Vendor and part number
Universal Human Reference RNA
Agilent p/n 740000
Universal Mouse Reference RNA
Agilent p/n 740100
Universal Rat Reference RNA
Agilent p/n 740200
Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
www.agilent.com
In This Book
This guide contains
information to run the
Two-Color
Microarray-Based Gene
Expression Analysis
protocol.
Agilent Technologies, Inc. 2007-2015
Version 6.9.1, August 2015
*G4140-90050*
G4140-90050
Revision B6
Agilent Technologies
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