agilent geneexpression two color v6.5

ATwoMicExp

Low

ProFor use w

Version 6

MicroaTechno

ResearProcedroarray-Based Gene ression Analysis

Input Quick Amp Labeling

tocol-Color gilent Technologies

ith Agilent Gene Expression oligo microarrays

.5, May 2010

rrays manufactured with Agilent SurePrint logy

ch Use Only. Not for use in Diagnostic ures.

2Notices Agilent Technologies, Inc. 2007-2010

No part oany formtronic stointo a forment andTechnoloStates an

ManuaG4140-90

EditionVersion 6

Printed in

Agilent T5301 SteSanta Cla

Warranty

erial contained in this nt is provided as is, and t to being changed,

notice, in future editions. to the maximum extent d by applicable law, isclaims all warranties, press or implied, with

o this manual and any ion contained herein, g but not limited to the warranties of merchant-nd fitness for a particular . Agilent shall not be r errors or for incidental quential damages in

ion with the furnishing, erformance of this docu-of any information d herein. Should Agilent

user have a separate agreement with warranty vering the material in this

nt that conflict with these e warranty terms in the agreement shall control.

252.227-7015 (Technical Data - Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Com-puter Software Documentation).

Safety Notices

CAUTION

A CAUTION notice denotes a haz-ard. It calls attention to an operat-ing 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.

WARNING

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly per-

AcknoAdobe

trademar

Pentium

Intel Corp

WindowsMicrosof

TechniTechnicaby contacServices wide salenumbersWeb sitewww.agi

or send agenomic

NoticeResearchprocedurf this manual may be reproduced in or by any means (including elec-rage and retrieval or translation eign language) without prior agree- written consent from Agilent gies, Inc. as governed by United d international copyright laws.

l Part Number050

.5, May 2010

USA

echnologies, Inc.vens Creek Rd ra, CA 95051 USA

The matdocumeis subjecwithout Further, permitteAgilent deither exregard tinformatincludinimplied ability apurposeliable foor conseconnectuse, or pment or containeand the written terms codocumeterms, thseparate

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cal Supportl product support may be obtained Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accor-dance with the terms of such license.

Restricted Rights LegendU.S. Government Restricted Rights. Soft-ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus-tomers. Agilent provides this customary commercial license in Software and techni-cal data pursuant to FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS

formed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated condi-tions are fully understood and met.

ting your local Agilent Support representative. Agilents world-s and support center telephone

can be obtained at the following : lent.com/chem/contactus

n email to: [email protected]

to Purchaser Use Only. Not for use in diagnostic es.

Two-Co

In this Guide...This document describes Agilent's recommended procedures for the prehybridAgilenmicroa

1 Before

This chsafety should

2 Proced

This chwash ausing t

templaozone-

protoclor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 3apter contains reference information related to the ol.4 Referen

This chapter contains instructions for quality assessment of te RNA and labeled cRNA, and steps to prevent related problems.

ce3 Supplem

This chparation and labeling of complex biological targets and ization, washing, scanning, and feature extraction of t 60-mer oligonucleotide microarrays for rray-based two-color gene expression analysis.

You Begin

apter contains information (such as procedural notes, information, required reagents and equipment) that you read and understand before you start an experiment.

ures

apter describes the steps to prepare samples, hybridize, nd scan gene expression microarrays, and to extract data he Agilent Feature Extraction Software.

ental Procedures

4Whats New in Version 6.5? SurePrint G3 (1x1M, 2x400K, 4x180K, and 8x60K)

Wha

UpdSca

Theice Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolated settings are provided for the Agilent Microarray nner.

hybridization procedure now includes the addition of an step.microarray formats are supported.

Stratagene Absolute RNA Nanoprep kit to purify labeled/amplified RNA is supported.

The cRNA hybridization input has been updated for the 1-pack and 2-pack microarray format.

The starting input requirement has been reduced to 10 ng for the 8-pack microarray format.

ts New in Version 6.0? The Agilent Low Input Quick Amp Labeling Kit is supported

in addition to the Agilent Quick Amp Labeling Kit.

An optional thermocycler protocol has been added.

Agilent Ozone-Barrier Slide Covers can be used to prevent ozone-related problems.

Two-Co

Content

1 Before

ProSafAgiReqReqOptReqOpt

2 Proced

Sample

SteSteSteSte

Hybrid

SteSte

SteSteSteSte

Scanni

SteStelor Microarray-Basedp 1. Add Triton X-102 to Gene Expression wash buffers 36p 2. Prewarm Gene Expression Wash Buffer 2 37p 3. Prepare the equipment 37p 4. Wash the microarray slides 39

ng and Feature Extraction 42

p 1. Scan the slides 42p 2. Extract data using Agilent Feature Extraction Software 45Step 3. Prepare the hybridization assembly 34

Microarray Wash 36 You Begin 7

cedural Notes 8ety Notes 9lent Oligo Microarray Kit Contents 10uired Equipment 11uired Reagents 12ional Equipment/Reagents 12uired Hardware and Software 13ional Software 13

ures 15

Preparation 17

p 1. Prepare Spike A Mix and Spike B Mix 19p 2. Prepare labeling reaction 23p 3. Purify the labeled/amplified RNA 27p 4. Quantify the cRNA 29

ization 31

p 1. Prepare the 10X Blocking Agent 31p 2. Prepare hybridization samples 32 Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 5

6 Two-Color Micro

Contents

3 Supplemental Procedures 51

Absolute RNA Nanoprep Purification 52

Step 1. Prepare the reagents 52Ste

Therm

SteSteSte

Quick A

SteSteSte

SteSteSte

Preven

SteSte

4 Refere

SupMicGenArrArrRelarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

p 1. Prepare for quality assessment 71p 2. Assess the quality using the Agilent 2100 Bioanalyzer 72p 3. Assess the quality using a NanoDrop Spectrophotometer 75

ting Ozone-Related Problems 76

p 1. Prepare the Stabilization and Drying Solution 77p 2. Wash with Stabilization and Drying Solution 78

nce 81

plemental User Guides 82roarray Handling Tips 83eral Microarray Layout and Orientation 84

ay/Sample tracking on a 4-pack array slide 87ay/Sample tracking on a 8-pack array slide 88ated Microarray Reagents 89Step 4. Quantify the cRNA 68

Quality Assessment of Template RNA and Labeled cRNA 70p 2. Purify the labeled/amplified RNA 53

ocycler Protocol 55

p 1. Program the thermocycler 55p 2. Synthesize cDNA from Total RNA 56p 3. Synthesize Fluorescent cRNA Synthesis in vitro 57

mp Labeling Kit Sample Preparation 58

p 1. Prepare Spike A Mix and Spike B Mix 60p 2. Prepare labeling reaction 63p 3. Purify the labeled/amplified RNA 66

Two-Color Microarray-Based Gene Expression AnalysisProtocol

1Before You Begin

N7Agilent TechnologiesProcedural Notes 8

Safety Notes 9

Agilent Oligo Microarray Kit Contents 10

Required Equipment 11

Required Reagents 12

Optional Equipment/Reagents 12

Required Hardware and Software 13

Optional Software 13

Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.

OTE Agilent cannot guarantee microarray performance and does not provide technical support to those who use non-Agilent protocols in processing Agilent's microarrays.

1 Before You Begin Procedural Notes

8

Procedural Notes

Determine the integrity of the input RNA for labeling and hybridization prio

To ppowwith

Mai

Whe

1 Tt

2 Mt

3 S

In gTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolr to use to increase the likelihood of a successful experiment.

revent contamination of reagents by nucleases, always wear der-free laboratory gloves, and use dedicated solutions and pipettors nuclease-free aerosol-resistant tips.

ntain a clean work area.

n preparing frozen reagent stock solutions for use:

haw the aliquot as rapidly as possible without heating above room emperature.

ix briefly on a vortex mixer, then centrifuge for 5 to 10 seconds to drive he contents off of walls and lid.

tore on ice or in a cold block until use.

eneral, follow Biosafety Level 1 (BL1) safety rules.

Before You Begin 1Safety Notes

Two-Co

Safety Notes

CAUTION Inspect the Stabilization and Drying Solution bottle for chips or cracks prior to use.

WAlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 9

Failure to do so may result in bottle breakage.

Wear appropriate personal protective equipment (PPE) when working in the laboratory.

RNING Cyanine 3-CTP and cyanine 5-CTP 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 Agilents 2X 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 Agilents 2X Hybridization Buffer.

Triton is harmful if swallowed. Risk of serious damage to eyes. Wear suitable PPE. Triton is a component of Agilents 2X 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.

Agilent 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.

1 Before You Begin Agilent Oligo Microarray Kit Contents

10

Agilent Oligo Microarray Kit Contents

Check the Agilent Web site at www.agilent.com/chem/dualmode for the most

NTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolup to date list of supported microarray designs.

Catalog microarray kits

One to eight microarrays printed on each 1-inch 3-inch glass slides, of a five slide kit.

DVD containing microarray design files in various file formats

Custom microarray kits

One to eight microarrays printed on each 1-inch 3-inch glass slide

Number of microarrays varies per kit and per order

OTE 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.

Before You Begin 1Required Equipment

Two-Co

Required Equipment

Description Vendor and part number

Agilent M

Hybridiz

Hybridiz 1 mic 2 mic 4 mic 8 mic

Hybridiz

HybridizChambe

Nucleas

Magnet

Magnet

Microce

NanoDro

Slide-sta

Circulati40C, 70

Clean fo

Ice buck

Pipetmaequivale

Powder-

Sterile, n

Vortex m

Timer

Nitrogenlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 11icroarray Scanner Agilent p/n G2565BA or G2565CA

ation Chamber, stainless Agilent p/n G2534A

ation Chamber gasket slidesroarray/slide, 5 slides/boxroarrays/slide, 5 slides/boxroarrays/slide, 5 slides/boxroarrays/slide, 5 slides/box

Agilent p/n G2534-60003Agilent p/n G2534-60002Agilent p/n G2534-60011Agilent p/n G2534-60014

ation oven; temperature set at 65C Agilent p/n G2545A

ation oven rotator for Agilent Microarray Hybridization rs

Agilent p/n G2530-60029

e-free 1.5 mL microfuge tubes Ambion p/n 12400 or equivalent

ic stir bar (2) Corning p/n 401435 or equivalent

ic stir plate (2) Corning p/n 6795-410 or equivalent

ntrifuge Eppendorf p/n 5417R or equivalent

p ND-1000 UV-VIS spectrophotometer NanoDrop p/n ND-1000 or equivalent

ining dish, with slide rack (3) Thermo Shandon p/n 121 or equivalent

ng water baths or heat blocks set to 37C, 65C, 80C, C, and 60C

rceps

et

n micropipettors, (P-10, P-20, P-200, P-1000) or nt

free gloves

uclease-free aerosol barrier pipette tips

ixer

purge box for slide storage

1 Before You Begin Required Reagents

12

Required Reagents

Optiona

Description Vendor and part number

Low Inp

RNA Sp

Gene Ex

Gene Ex

DNase/

Milli-Q w

RNeasy

100% Et

Descrip

2100 Bio

Stabilization and Drying Solution Agilent p/n 5185-5979

Ozone-B

Acetoni

Absolut

Thermoc

PCR 96-Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolarrier Slide Cover Agilent p/n G2505-60550

trile Sigma p/n 271004-1L

ely RNA Nanoprep Kit Stratagene p/n 4000753

ycler

well plate or 0.2 mL PCR tubesRNA 6000 Nano Assay Kit (RNA Series II Kit) Agilent p/n 5067-1511

Slide box Corning p/n 07201629l Equipment/Reagents

ut Quick Amp Labeling Kit, Two-Color Agilent p/n 5190-2306

ike-In Kit, Two-Color Agilent p/n 5188-5279

pression Hybridization Kit Agilent p/n 5188-5242

pression Wash Buffer Kit Agilent p/n 5188-5327

RNase-free distilled water Invitrogen p/n 10977-015

ater or equivalent

Mini Kits (50 columns or 250 columns) Qiagen p/n 74104 or 74106

hanol Amresco p/n E193

tion Vendor and part number

analyzer Agilent p/n G2938A

Before You Begin 1Required Hardware and Software

Two-Co

Required Hardware and Software

Optiona

Description

Pentium

Agilents

2 GB RA

40 GB a

Window

Feature

Internet

Adobe A

Descrip

GeneSplor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 13l Software

III 1.5 GHz or higher (Pentium IV 2.0 GHz or higher recommended)

Scan Control software, version A.7.0.1 for the B Scanner or A.8.4.1 for the C Scanner

M (4 GB recommended for 64-bit operating systems)

vailable disk space (if saving images and results files locally)

s 2000 with SP2 or later (fully tested on SP4), Windows XP SP2

Extraction software 9.5 or later

Explorer 6.0 or later

crobat Reader 4.0 or later

tion

ring GX 10.0 or later

1 Before You Begin Optional Software

14 Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol


2Procedures

Agilen3- andand coand ar15Agilent Technologies

Sample Preparation 17

Step 1. Prepare Spike A Mix and Spike B Mix 19

Step 2. Prepare labeling reaction 23

Step 3. Purify the labeled/amplified RNA 27

Step 4. Quantify the cRNA 29

Hybridization 31

Step 1. Prepare the 10X Blocking Agent 31

Step 2. Prepare hybridization samples 32

Step 3. Prepare the hybridization assembly 34

Microarray Wash 36

Step 1. Add Triton X-102 to Gene Expression wash buffers 36

Step 2. Prewarm Gene Expression Wash Buffer 2 37

Step 3. Prepare the equipment 37

Step 4. Wash the microarray slides 39

Scanning and Feature Extraction 42

Step 1. Scan the slides 42

Step 2. Extract data using Agilent Feature Extraction Software 45

t's Two-Color Microarray-based Gene Expression Analysis uses cyanine cyanine 5-labeled targets to measure gene expression in experimental ntrol samples. Figure 1 is a standard workflow for sample preparation ray hybridization design.

2 Procedures

16 Two-Color Micro

Figure

Template Total or poly A+ RNA with Spike-In

* Samarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

1 Workflow for sample preparation and array processing.

cDNA synthesis*

cRNA synthesis and amplification*

cRNA purification*

Preparation of hybridization sample

17-hour hybridization (65C)

Wash

Scan

Feature extraction

ples can be stored frozen at -80C after these steps, if needed.

Procedures 2Sample Preparation

Two-Co

Sample Preparation

Agilent's Low Input Quick Amp Labeling Kit generates fluorescent cRNA (comp200 ngprocesamplifCTP. Athe us

NOTE For optirefer toguidanclor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 17limentary RNA) with a sample input RNA range between 10 ng and of total RNA or a minimum of 5 ng of poly A+ RNA for two-color sing. The method uses T7 RNA polymerase, which simultaneously ies target material and incorporates cyanine 3- or cyanine 5-labeled mplification is typically at least a 100-fold from total RNA to cRNA with e of this kit.

mal performance, use high quality, intact template total or poly A+ RNA. Please Quality Assessment of Template RNA and Labeled cRNA on page 70 for general e and procedural recommendations on quality assessment of template RNA.

2 Procedures Sample Preparation

18 Two-Color Micro

Figure array-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

2 Schematic of amplified cRNA procedure. Generation of cRNA for a two-color microarray experiment is shown. When you generate targets for a one-color microarray experiment, only the Cy3-labeled B sample is produced and hybridized.

Procedures 2Step 1. Prepare Spike A Mix and Spike B Mix

Two-Co

Step 1. Prepare Spike A Mix and Spike B Mix

(Time required: ~0.5 hours)

Refer tin-depspike mand cawww.a

1 Equ

2 Vigo

3 Hea

4 Brieto ooccu

Table differelabel SschemSpike StatistExtracsoftwahave tolor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 19o the protocol on Agilent RNA Spike-In Kit (publication 5188-5928) for th instructions and troubleshooting advice on how to use two-color

ixes. This protocol is available with the Two-Color RNA Spike-In Kit n also be downloaded from the Agilent Web site at gilent.com/chem/dnamanuals-protocols.

ilibrate water baths to 37C, 65C, 80C, 40C and 70C.

rously mix the stock solutions on a vortex mixer.

t at 37C for 5 minutes, and mix on a vortex mixer once more.

fly spin in a centrifuge to drive contents to the bottom of the tube prior pening. Settlement of the solution on the sides or lid of the tubes may r during shipment and storage.

1 provides the dilutions of Spike A Mix and Spike B Mix for four nt starting sample inputs of total RNA or 5 ng of poly A mRNA. Always pike A Mix with cyanine 3 and Spike B Mix with cyanine 5. The dilution e for Spike A Mix with cyanine 3 is the same as the dilution scheme for B Mix with cyanine 5. The output of the Agilent SpikeIns LogRatio ics table in the QC report, generated when using Agilent Feature tion Software 9.5 or later, uses this orientation. It is built into the re code and cannot be set by the user. If the polarity is flipped, you will manually adjust the output.

2 Procedures Step 1. Prepare Spike A Mix and Spike B Mix

20 Two-Color Micro

For exRNA s

1 Cre

a LD

e St

f I

g A

h Mt

Table 1 Dilutions of Spike A Mix for cyanine 3 and Spike B Mix for cyanine 5 labeling

Starting Amount of RNA Serial Dilution Spike Mix Volume to be used

Total RNA (ng

10

25

50

100

200

NOTE Use RNpipette array-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

pin briefly in a centrifuge to separate contents to the bottom of the ube.

nto the First Dilution tube, put 2 L of Spike A Mix stock.

dd 38 L of Dilution Buffer provided in the Spike-In kit (1:20).

ix thoroughly on a vortex mixer and spin down quickly to collect all of he liquid at the bottom of the tube. This tube contains the First Dilution.b Mix the thawed Spike A Mix vigorously on a vortex mixer.

c Heat at 37C in a circulating water bath for 5 minutes.

d Mix the Spike A Mix tube vigorously again on a vortex mixer.ample, to prepare the Spike A Mix dilution appropriate for 25 ng of total tarting sample:

ate the First Dilution:

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix First ilution.

in each labeling reaction (L)

)PolyARNA (ng)

First Second Third Fourth

1:20 1:40 1:16 1:20 2

1:20 1:40 1:16 1:8 2

1:20 1:40 1:16 1:4 2

1:20 1:40 1:16 1:2 2

1:20 1:40 1:16 2

5 1:20 1:40 1:16 1:2 2

ase-free microfuge tubes and tips. Make sure you dispense at least 2 L with a to ensure accuracy.

Procedures 2Step 1. Prepare Spike A Mix and Spike B Mix

Two-Color Microarray-Based

2 Create the Second Dilution:

a Label a new sterile 1.5 mL microcentrifuge tube Spike A Mix Second Dilution.

b I

c A

d MtD

3 Cre

a LD

b I

c A

d Ml

4 Cre

a LD

b ID

c MtD

5 AddTabQuireac Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 21

nto the Second Dilution tube, put 2 L of First Dilution.

dd 78 L of Dilution Buffer (1:40).

ix thoroughly on a vortex mixer and spin down quickly to collect all of he liquid at the bottom of the tube. This tube contains the Second ilution.

ate the Third Dilution:

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix Third ilution.

nto the Third Dilution tube, put 2 L of Second Dilution.

dd 30 L of Dilution Buffer (1:16).

ix thoroughly on a vortex mixer and spin down quickly to collect all the iquid at the bottom of the tube. This tube contains the Third Dilution.

ate the Fourth Dilution:

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix Fourth ilution.

nto the Fourth Dilution tube, add 4 L of Third Dilution to 28 L of ilution Buffer for the Fourth Dilution (1:8).

ix thoroughly on a vortex mixer and spin down quickly to collect all of he liquid at the bottom of the tube. This tube contains the Fourth ilution (now at a 102,400-fold final dilution).

2 L of Fourth Dilution to 25 ng of sample total RNA as listed in le 1 and continue with cyanine 3 labeling using the Agilent Low Input ck Amp Kit protocol as described in Step 2. Prepare labeling tion on page 23.

2 Procedures Step 1. Prepare Spike A Mix and Spike B Mix

22 Two-Color Micro

Storage of Spike Mix dilutions

Store the Agilent RNA Spike-In Kit, Two-Color at 70C to 80C in a non-defrosting freezer for up to 1 year from the date of receipt.

The firstoredfreeze/

After uarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

st dilution of the Agilent Spike Mix (A or B) positive controls can be up to 2 months in a non-defrosting freezer at 70C to 80C and thawed up to eight times.

se, discard the second, third and fourth dilution tubes.

Procedures 2Step 2. Prepare labeling reaction

Two-Co

Step 2. Prepare labeling reaction


For eadilutedinvolvdividesamplemaster

1 AddmicconRNAstor

2 Pre5-C

3 Addvolu

NOTE The statotal RNformatsmicroarlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 23ch assay, make sure that the volume of the total RNA sample plus RNA spike-in controls does not exceed 3.5 L. Because the 1x reaction

es volumes of less than 1 L, prepare components in a master mix and into the individual assay tubes in volumes >1 L. When preparing 4 s, use the 5x master mix. When preparing 8 samples, use the 10x mix.

10 to 200 ng of total RNA or 5 ng polyA RNA to a 1.5-mL rocentrifuge tube in a final volume of 1.5 L. If samples are centrated, dilute with water until 10 to 200 ng of total or 5 ng of polyA is added in a 1.5 L volume. Dilute the total RNA just prior to use and

e the total RNA at concentrations over 100 ng/L.

pare tubes for both Spike A Mix/cyanine 3-CTP and Spike B Mix/cyanine TP dyes.

2 L of diluted Spike Mix to each tube. Each tube now contains a total me of 3.5 L.

rting input for the Low Input Quick Amp Labeling Kit ranges from 10 ng to 200 ng of A. For best results, start with at least 25 ng of total RNA for the 4-pack and 8-pack , and 50 ng of total RNA for the 1-pack and 2-pack formats. For the 8-pack ray format, as little as 10 ng of total RNA can be used to generate high quality data.

2 Procedures Step 2. Prepare labeling reaction

24 Two-Color Micro

4 Prepare and add T7 Promoter Primer:

a Mix the T7 Promoter Primer and water to prepare the T7 Promoter Primer Master Mix as listed in Table 2.

b Att

c Di

d P

5 Preaderesumicroo

6 Pre

a Ilt

TAi

Table 2

Compon

T7 Prom

Nucleas

Total Voarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

dd 1.8 L of T7 Promoter Primer Mix to the tube that contains 3.5 L of otal RNA and diluted RNA spike-in controls. Each tube now contains a otal volume of 5.3 L.

enature the primer and the template by incubating the reaction at 65C n a circulating water bath for 10 minutes.

lace the reactions on ice and incubate for 5 minutes.

warm the 5X first strand buffer at 80C for 3 to 4 minutes to ensure quate resuspensions of the buffer components. For optimal spension, briefly mix on a vortex mixer and spin the tube in a

rocentrifuge to drive down the contents from the tube walls. Keep at m temperature until needed.

pare and add cDNA Master Mix:

mmediately prior to use, add the components for cDNA Master Mix isted in Table 3, use a pipette to gently mix, and keep at room emperature.

he AffinityScript RNase Block mix is a blend of enzymes. Keep the ffinityScript RNase Block mix on ice and add to the cDNA Master Mix

mmediately prior to use.

T7 Promoter Primer Mix

ent Volume (L) per reaction

Volume (L) per 5 reaction

Volume (L) per 10 reactions

oter Primer (green cap) 0.8 4 8

e-free water (white cap) 1 5 10

lume 1.8 9 18

Procedures 2Step 2. Prepare labeling reaction


b B

u

d I

e M

f M

g Sf

Stopping Point If you 80C

7 Pre

a Itr

Tpu

Table 3 cDNA Master Mix

Component Volume (L) Volume (L) Volume (L)

5X First

0.1 M DT

10 mM d

AffinityS

Total Vo

N Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 25

ove samples to ice. Incubate for 5 minutes.

pin samples briefly in a microcentrifuge to drive down tube contents rom the tube walls and lid.

do not immediately continue to the next step, store the samples at .

pare and add Transcription Master Mix:

mmediately prior to use, gently mix the components listed in Table 4 in he order indicated for the Transcription Master Mix by pipetting at oom temperature.

he T7 RNA polymerase blend is a blend of enzymes. Keep the T7 RNA olymerase on ice and add to the Transcription Master Mix just before se.15 minutes.

OTE Incubation at 70C inactivates the AffinityScript enzyme.dd 4.7 L of cDNA Master Mix to each sample tube and mix by pipetting p and down. Each tube now contains a total volume of 10 L.

ncubate samples at 40C in a circulating water bath for 2 hours.

ove samples to a 70C circulating water bath and incubate for co

c Ariefly spin each sample tube in a microcentrifuge to drive down the ntents from the tube walls and the lid.

per reaction per 5 reaction per 10 reactions

Strand Buffer (green cap) 2 10 20

T (white cap) 1 5 10

NTP mix (green cap) 0.5 2.5 5

cript RNase Block Mix (violet cap) 1.2 6 12

lume 4.7 23.5 47

2 Procedures Step 2. Prepare labeling reaction

26 Two-Color Micro

b Ap

c I

Stopping Point If you 80C

Table 4 Transcription Master Mix

Component Volume (L) per Volume (L) per Volume (L) per

Nucleas

5X Trans

0.1 M DT

NTP mix

T7 RNA

Cyanine

Total Voarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocoldd 6 L of Transcription Master Mix to each sample tube. Gently mix by ipetting. Each tube now contains a total volume of 16 L.

ncubate samples in a circulating water bath at 40C for 2 hours.

do not immediately continue to the next step, store the samples at .

reaction 5 reaction 10 reactions

e-free water (white cap) 0.75 3.75 7.5

cription Buffer (blue cap) 3.2 16 32

T (white cap) 0.6 3 6

(blue cap) 1 5 10

Polymerase Blend (red cap) 0.21 1.05 2.1

3-CTP or cyanine 5-CTP 0.24 1.2 2.4

lume 6 30 60

Procedures 2Step 3. Purify the labeled/amplified RNA

Two-Co

Step 3. Purify the labeled/amplified RNA


1 Add100

2 Add

3 Addpipe

4 Tra2 m13,0

5 Trabuff4Ccoll

6 Add4Ccoll

7 If anRNe

NOTE Make sbefore y

CAUTION Do not lor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 27discard the final flow-through in the next step. It contains the cRNA sample.4C for 30 seconds at 13,000 rpm to remove any remaining traces of buffer RPE. Discard this collection tube and use a fresh tube to elute the cleaned cRNA sample. 84 L of nuclease-free water to your cRNA sample, for a total volume of L.

350 L of Buffer RLT and mix well by pipetting.

250 L of ethanol (96% to 100% purity) and mix thoroughly by tting. Do not centrifuge.

nsfer the 700 L of the cRNA sample to an RNeasy mini column in a L collection tube. Centrifuge the sample at 4C for 30 seconds at 00 rpm. Discard the flow-through and collection tube.

nsfer the RNeasy column to a new collection tube and add 500 L of er RPE (containing ethanol) to the column. Centrifuge the sample at for 30 seconds at 13,000 rpm. Discard the flow-through. Re-use the ection tube.

another 500 L of buffer RPE to the column. Centrifuge the sample at for 60 seconds at 13,000 rpm. Discard the flow-through and the ection tube.

y buffer RPE remains on or near the frit of the column, transfer the asy column to a new 1.5 mL collection tube and centrifuge the sample at

ure that ethanol was added to the RPE buffer as specified in the Qiagen manual ou continue.Qiagens RNeasy mini spin columns are recommended for purification of the amplified cRNA samples.

If sample concentration causes difficulty, you can use the Stratagene Absolutely RNA Nanoprep kit as an alternative. See Absolute RNA Nanoprep Purification on page 52.

2 Procedures Step 3. Purify the labeled/amplified RNA

28 Two-Color Micro

8 Elute the cleaned cRNA sample by transferring the RNeasy column to a new 1.5 mL collection tube. Add 30 L RNase-free water directly onto the RNeasy filter membrane. Wait 60 seconds, then centrifuge at 4C for 30 s

9 MaiRNearray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

econds at 13,000 rpm.

ntain the cRNA sample-containing flow-through on ice. Discard the asy column.

Procedures 2Step 4. Quantify the cRNA

Two-Co

Step 4. Quantify the cRNA

Quantitate cRNA using NanoDrop ND-1000 UV-VIS Spectrophotometer versio

1 Star

2 Clic

3 Befosam

4 Loa

5 Oncthe

6 Makthe

7 Blancan

8 Cleaof tnam

9 Prinvalu

C

R

c

CAUTION Failurepossibilor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 29easure the sample.

t the results. If printing the results is not possible, record the following es:

yanine 3 or cyanine 5 dye concentration (pmol/L)

NA absorbance ratio (260 nm/280 nm)

RNA concentration (ng/L)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 remn 3.2.1.

t the NanoDrop software.

k the Microarray Measurement tab.

re initializing the instrument as requested by the software, clean the ple loading area with nuclease-free water.

d 1.0 to 2.0 L of nuclease-free water to initialize. Then click OK.

e the instrument has initialized, select RNA-40 as the Sample type (use drop down menu).

e sure the Recording button is selected. If not, click Recording so that readings can be recorded, saved, and printed.

k the instrument by pipetting 1.0 to 2.0 L of nuclease-free water (this be the same water used to initialize the instrument) and click Blank.

n the sample loading area with a laboratory wipe. Pipette 1.0 to 2.0 L he sample onto the instrument sample loading area. Type the sample e in the space provided and click Measure.

to engage recording causes measurements to be overwritten, with no lity of retrieval.

2 Procedures Step 4. Quantify the cRNA

30 Two-Color Micro

10 Determine the yield and specific activity of each reaction as follows:

a Use the concentration of cRNA (ng/L) to determine the g cRNA yield as follows:

8-pack

NOTE Please generalcRNA.array-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

0.825 6

refer to Quality Assessment of Template RNA and Labeled cRNA on page 70 for guidance and procedural recommendations on quality assessment of labeled b Use the concentrations of cRNA (ng/L) and cyanine 3 or cyanine 5 (pmol/L) to determine the specific activity as follows:

11 Examine the yield and specific activity results. See Table 5 for the recommended cRNA yields and specific activities for hybridization.

Table 5 Recommended Yields and Specific Activity

Microarray format Yield (g) Specific Activity (pmol Cy3 or Cy5 per g cRNA)

1-pack 2.5 6

2-pack 1.875 6

4-pack 0.825 6

(Concentration of cRNA) 30 L (elution volume)1000

----------------------------------------------------------------------------------------------------------------------------------------- g of cRNA=

Concentration of Cy3 or Cy5Concentration of cRNA

----------------------------------------------------------------------------- 1000 pmol Cy3 per g cRNA=

Procedures 2Hybridization

Two-Co

Hybridization

Nlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 31Step 1. Prepare the 10X Blocking Agent

1 Add 500 L of nuclease-free water to the vial containing lyophilized 10X Blocking Agent supplied with the Agilent Gene Expression Hybridization Kit, or add 1250 L of nuclease-free water to the vial containing lyophilized large volume 10X Blocking Agent (Agilent p/n 5188-5281).

2 Mix by gently vortexing. If the pellet does not go into solution completely, heat the mix for 4 to 5 minutes at 37C.

3 Drive down any material adhering to the tube walls or cap by centrifuging for 5 to 10 seconds.

OTE 10X Blocking Agent can be prepared in advance and stored at 20C for up to 2 months. After thawing, repeat the vortexing and centrifugation procedures before use.

2 Procedures Step 2. Prepare hybridization samples

32

Step 2. Prepare hybridization samples

1 Equilibrate water bath to 60C.

2 Forbelo

T

T

3 Mix

NOTE For 1-paamounteach m

Table 6

Compon

cyanine

cyanine

10X Bloc

Nucleas

25X Frag

Total VoTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol each microarray, add each of the components as indicated in the tables w to a 1.5 mL nuclease-free microfuge tube:

able 6 for 1-pack or 2-pack microarray formats

able 7 for 4-pack or 8-pack microarray formats

well but gently on a vortex mixer.

ck and 2-pack microarrays, if you did not generate enough labeled cRNA, add the of labeled cRNA to the fragmentation mix such that the same amount is used for icroarray within the same experiment (at least 1.65 g).

Fragmentation mix for 1-pack or 2-pack microarray formats

ents Volume/Mass1-pack microarrays

Volume/Mass2-pack microarrays

3-labeled, linearly amplified cRNA 2.5 g 1.875 g

5-labeled, linearly amplified cRNA 2.5 g 1.875 g

king Agent 50 L 25 L

e-free water bring volume to 240 L bring volume to 120 L

mentation Buffer 10 L 5 L

lume 250 L 125 L

Procedures 2Step 2. Prepare hybridization samples


4 Incu

5 Imm

6 Add8-pafrag

7 Mixnot

8 Spinto d

Use

9 Plac

Refer tsafely

Table 7 Fragmentation mix for 4-pack or 8-pack microarray formats

Components Volume/Mass4-pack microarrays

Volume/Mass8-pack microarrays

cyanine

cyanine

10X Bloc

Nucleas

25X Frag

Total Vo

CAUTION Do not adding

Table 8

Compon Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 33

well by careful pipetting. Take care to avoid introducing bubbles. Do mix on a vortex mixer; mixing on a vortex mixer introduces bubbles.

for 1 minute at room temperature at 13,000 rpm in a microcentrifuge rive the sample off the walls and lid and to aid in bubble reduction.

immediately. Do not store.

e sample on ice and load onto the array as soon as possible.

o Microarray Handling Tips on page 85 for information on how to handle microarrays.cRNA from Fragmentation Mix 250 L 125 L 55 L 25 L

2x GEx Hybridization Buffer HI-RPM 250 L 125 L 55 L 25 Lbate at 60C for exactly 30 minutes to fragment RNA.

ediately cool on ice for one minute.

2x GEx Hybridization Buffer HI-RPM to the 1-pack, 2-pack, 4-pack, and ck microarray formats at the appropriate volume to stop the mentation reaction. See Table 8.

3-labeled, linearly amplified cRNA 825 ng 300 ng

5-labeled, linearly amplified cRNA 825 ng 300 ng

king Agent 11 L 5 L

e-free water bring volume to 52.8 L bring volume to 24 L

mentation Buffer 2.2 L 1 L

lume 55 L 25 L

incubate sample in the next step for more than 30 minutes. Cooling on ice and the 2x Hybridization Buffer will stop the fragmentation reaction.

Hybridization mix

Volumes per hybridization

ents 1-pack 2-pack 4-pack 8-pack

2 Procedures Step 3. Prepare the hybridization assembly

34

Step 3. Prepare the hybridization assembly

Refer to the Agilent Microarray Hybridization Chamber User Guide (G2534disassavailaband cawww.a

2 Slowgask

3 Slowthatis fa

NOTE Place ubarcodethe corr

CAUTION Do not liquid t

CAUTION When ythat theTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolly place an array active side down onto the SureHyb gasket slide, so the Agilent-labeled barcode is facing down and the numeric barcode cing up. Make sure the sandwich-pair is properly aligned.

ou lower the microarray slide on top of the SureHyb gasket slide, make sure two slides are parallel at all times.Table 9 Hybridization Sample

Volumes per hybridization

Components 1-pack 2-pack 4-pack 8-pack

Volume Prepared 500 L 250 L 110 L 50 L

Volume to Hybridize 490 L 240 L 100 L 40 Lly dispense the volume of hybridization sample (see Table 9) onto the et well in a drag and dispense manner.

nused gasket wells in the multi-pack array format at the far end opposite the . In the unused wells, maintain the appropriate volume of 1x hybridization buffer for esponding format design.

let the pipette tip or the hybridization solution touch the gasket walls. When ouches the gasket wall, the likelihood of gasket leakage greatly increases.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. Ensure that the gasket slide is flush with the chamber base and is not ajar.-90001) for in-depth instructions on how to load slides, assembly and embly of chambers, as well as other helpful tips. This user guide is le with the Agilent Microarray Hybridization Chamber Kit (G2534A) n also be downloaded from the Agilent Web site at gilent.com/chem/dnamanuals-protocols.

Procedures 2Step 3. Prepare the hybridization assembly

Two-Co

CAUTION Do not drop the array slide onto the gasket. Doing so increases the chances of samples mixing between gasket wells.

CA

Nlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 354 Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces.

5 Hand-tighten the clamp onto the chamber.

6 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.

7 Place assembled slide chamber in rotisserie in a hybridization oven set to 65C. Set your hybridization rotator to rotate at 10 rpm when using 2x GEx Hybridization Buffer HI-RPM.

8 Hybridize at 65C for 17 hours.

UTION 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.

OTE 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.

2 Procedures Microarray Wash

36

Microarray Wash

Step 1.

This st

The adreduceExpreopened

Do thi

1 Operem

2 Usebuff

3 Repthor

4 Carwith

5 Probee

Tritonfinal dbufferTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) ProtocolAdd Triton X-102 to Gene Expression wash buffers

ep is optional but highly recommended.

dition of 0.005% Triton X-102 to the Gene Expression wash buffers s the possibility of array wash artifacts. Add the Triton X-102 to Gene

ssion wash buffer 1 and 2 when the cubitainer of wash buffer is first .

s step to both Gene Expression wash buffer 1 and 2 before use.

n the cardboard box with the cubitainer of wash buffer and carefully ove the outer and inner caps from the cubitainer.

a pipette to add 2 mL of the provided 10% Triton X-102 into the wash er in the cubitainer.

lace the original inner and outer caps and mix the buffer carefully but oughly by inverting the container 5 to 6 times.

efully remove the outer and inner caps and install the spigot provided the wash buffer.

minently label the wash buffer box to indicate that Triton X-102 has n added and indicate the date of addition.

X-102 can be added to smaller volumes of wash buffer as long as the ilution of the 10% Triton X-102 is 0.005% in the Gene Expression wash solution.

Procedures 2Step 2. Prewarm Gene Expression Wash Buffer 2

Two-Co

Step 2. Prewarm Gene Expression Wash Buffer 2

Warm the Gene Expression Wash Buffer 2 to 37C as follows:

1 Disp100solu

2 Tighbefobox

Step 3.

Alway

Aceton

Wash sexperiaceton

3 Fill the staining dish with 100% acetonitrile.

4 Tur(me

5 Was

6 Disc

WARNING Conduclor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 37n on the magnetic stir plate and adjust the speed to a setting of 4 dium speed).

h for 5 minutes.

ard the acetonitrile as is appropriate for your site.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.itrile wash

taining dishes, racks and stir bars that were used in previous ments with the Agilent Stabilization and Drying Solution with itrile to remove any remaining residue.

t acetonitrile washes in a vented fume hood.Designate and dedicate dishes to two-color experiments. The acetonitrile wash is only necessary if the staining dishes, racks and stir bars were used in previous experiments with the Agilent Stabilization and Drying Solution. Otherwise proceed to Milli-Q water wash on page 38.ense 1000 mL of Gene Expression Wash Buffer 2 directly into a sterile 0-mL bottle. Repeat until you have enough prewarmed Wash Buffer 2 tion for your experiment.

tly cap the 1000-mL bottle and place in a 37C water bath the night re washing arrays. Alternatively, remove the plastic cubitainer from the

and place it in a 37C water bath the night before washing the arrays.

Prepare the equipment

s use clean equipment when doing the hybridization and wash steps.

2 Procedures Step 3. Prepare the equipment

38 Two-Color Micro

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 below.

CAarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) ProtocolMilli-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.

UTION 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.

Procedures 2Step 4. Wash the microarray slides

Two-Co

Step 4. Wash the microarray slides

Table

1 Comat r

2 Placslidtem

3 Placaddwas

4 Remwhe

NOTE The microarray wash procedure for Agilents two-color platform must be done in environin your ozone l

NOTE When scontain

Table 1

Disasse

1st wash

2nd waslor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 39

freely.10 lists the wash conditions for the wash procedure.

pletely fill slide-staining dish #1 with Gene Expression Wash Buffer 1 oom temperature.

e a slide rack into slide-staining dish #2. Add a magnetic stir bar. Fill e-staining dish #2 with enough Gene Expression Wash Buffer 1 at room perature to cover the slide rack. Place this dish on a magnetic stir plate.

e the empty dish #3 on the stir plate and add a magnetic stir bar. Do not the prewarmed (37C) Gene Expression Wash Buffer 2 until the first h step has begun.

ove one hybridization chamber from incubator and record time. Record ther bubbles formed during hybridization and if all bubbles are rotating

ments where ozone levels are 5 ppb or less. If ozone levels are between 5 to 10 ppb laboratory, use the Agilent Ozone Barrier Slide Cover (described in this topic). If evels exceed 10 ppb seePreventing Ozone-Related Problems on page 76.

etting up the apparatus for the washes, be sure to do so near the water bath ing the pre-warmed Wash 2 solutions.

0 Wash conditions

Dish Wash Buffer Temperature Time

mbly 1 GE Wash Buffer 1 Room temperature

2 GE Wash Buffer 1 Room temperature

1 minute

h 3 GE Wash Buffer 2 Elevated temperature

1 minute

2 Procedures Step 4. Wash the microarray slides

40 Two-Color Micro

5 Prepare the hybridization chamber disassembly.

a Place the hybridization chamber assembly on a flat surface and loosen the thumbscrew, turning counterclockwise.

b S

c Wbns

a S

b G

c L

d Rstb

Morslid

7 RepForyiel

8 Whedish

9 Dur37

10 TraWas

11 Slowtake

NOTE The elethe rooarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

h the sandwich completely submerged in Gene Expression Wash fer 1, pry the sandwich open from the barcode end only:

lip one of the blunt ends of the forceps between the slides.

ently turn the forceps upwards or downwards to separate the slides.

et the gasket slide drop to the bottom of the staining dish.

emove the microarray slide and place into slide rack in the lide-staining dish #2 containing Gene Expression Wash Buffer 1 at room emperature. Minimize exposure of the slide to air. Touch only the arcode portion of the microarray slide or its edges!

e effort is needed to separate the 4x44K than the 1x44K sandwiched es.

eat step 4 through step 6 for up to seven additional slides in the group. uniform washing, do up to a maximum of eight disassembly procedures ding eight microarray slides.

n all slides in the group are placed into the slide rack in slide-staining #2, stir using setting 4 for 1 minute.

ing this wash step, remove Gene Expression Wash Buffer 2 from the C water bath and pour into the slide-staining dish #3.

nsfer slide rack to slide-staining dish #3 containing Gene Expression h Buffer 2 at elevated temperature. Stir using setting 4 for 1 minute.

ly remove the slide rack minimizing droplets on the slides. It should 5 to 10 seconds to remove the slide rack.

vated temperature of the second wash step is usually around 31C due to cooling by m temperature dish and the rack of arrays.d Without letting go of the slides, submerge the array-gasket sandwich into slide-staining dish #1 containing Gene Expression Wash Buffer 1.

6 WitBuflide off the clamp assembly and remove the chamber cover.

ith gloved fingers, remove the array-gasket sandwich from the chamber ase by grabbing the slides from their ends. Keep the microarray slide umeric barcode facing up as you quickly transfer the sandwich to lide-staining dish #1.

Procedures 2Step 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 Genpre

14 Put

ItotG

Figure

Iw

15 Scaon snitr

NOTE Use fre Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 41

3 Inserting the ozone-barrier slide cover

n environments in which the ozone level is below 5 ppb, put the slides ith Agilent barcode facing up in a slide holder.

n slides immediately to minimize the impact of environmental oxidants ignal intensities. If necessary, store slides in orange slide boxes in a ogen purge box, in the dark.

sh Gene Expression Wash Buffer 1 and 2 for each wash group (up to 8 slides).e Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 -warmed to 37C.

the slides in a slide holder so that the Agilent barcode faces up:

n environments in which the ozone level exceeds 5 ppb, immediately put he slides with Agilent barcode facing up in a slide holder with an zone-barrier slide cover on top of the array as shown in Figure 3. Refer o the Agilent Ozone-Barrier Slide Cover User Guide (p/n 2505-90550), included with the slide cover, for more information.

2 Procedures Scanning and Feature Extraction

42

Scanning and Feature Extraction

Step 1.

Agilen

1 Putinto

2 In tfor

3 ForAgi

4 ForAgi

If yoConhttpPagCon

5 Veri

Table 1

Dye cha

Scan re

Scan re

TiffTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) ProtocolScan the slides

t C Scanner Settings

assembled slide holders with or without the ozone-barrier slide cover the scanner carousel.

he Scan Control main window, choose the slot number of the first slide Start Slot and the slot number for the last slide for End Slot.

1x244K, 2x105K, 4x44K and 8x15K microarrays, select Profile lentHD_GX_2Color.

1x1M, 2x400K, 4x180K and 8x60K microarrays, select Profile lentG3_GX_2Color.

u are unable to find the profile AgilentG3_GX_2Color from the Scan trol program, download the profile from s://www.genomics.agilent.com/GenericA.aspx?PageType=Custom&Sub

eType=Custom&PageID=2074. To import the profile into the Scan trol program, click Tools > Profile Editor and select Import.

fy scan settings for two-color scans. See Table 11 and Figure 4.

1 C Scanner Scan Settings

For 1x244K, 2x105K, 4x44K and 8x15K HD Microarray Formats

For 1x1M, 2x400K, 4x180K and 8x60K G3 Microarray Formats

nnel Red&Green Red&Green

gion Scan Area (61 x 21.6 mm) Scan Area (61 x 21.6 mm)

solution (m) 5 3

20 bit 20 bit

Procedures 2Step 1. Scan the slides

Two-Co

6 Veri

7 In tof t

Agilen

The Aguse th

1 Putinto

2 Veri

Figure 4 Agilent Scan Conlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 43fy that the Scanner status in the main window says Scanner Ready.

he Scan Control main window, click Scan Slot m-n where m is the slot he first slide, and n is the slot for the last slide.

t B Scanner Settings

ilent B Scanner does not support G3 microarrays. For G3 microarrays, e Agilent C Scanner.

assembled slide holders with or without the ozone-barrier slide cover the scanner carousel.

fy the scanner settings in Table 12.

trol dialog box shown in the HD Profile

2 Procedures Step 1. Scan the slides

44 Two-Color Micro

To cpop

3 Sele

P

P

4 Veri

5 Clicreprep

Table 12 B Scanner Scan Settings

For 1x244K, 2x105K Formats For 4x44K, 8x15K Formats

Scan reg

Scan res

5m sca

eXtende

Dye cha

Green P

Red PMarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

hange any settings, click Settings > Modify Default Settings. A window s up from which you can change the settings.

ct settings for the automatic file naming

refix 1 is set to Instrument Serial Number

refix 2 is set to Array Barcode

fy that the Scanner status in the main window says Scanner Ready.

k Scan Slot m-n on the Scan Control main window where the letter m resents the Start slot where the first slide is located and the letter n resents the End slot where the last slide is located.

ion Scan Area (61 x 21.6 mm) Scan Area (61 x 21.6 mm)

olution (m) 5 5

nning mode Single Pass Single Pass

d Dynamic range (selected)

nnel Red&Green Red&Green

MT 100% XDR Hi 100%XDR Lo 10%

T 100% XDR Hi 100%XDR Lo 10%

Procedures 2Step 2. Extract data using Agilent Feature Extraction Software

Two-Co

Step 2. Extract data using Agilent Feature Extraction Software

Featurextracexpressoftwawww.a

After gFeatur

1 Ope

To gto t

2 Add

a CP

b BO

Thefor

Ff

FdT

Tt

3 Set

a S

b I

c Is

d Ftlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 45e Extraction is the process by which information from probe features is ted from microarray scan data, allowing researchers to measure gene sion in their experiments. To get the most recent Feature Extraction re for gene expression, go to the Agilent Web site at gilent.com/chem/fe.

enerating the microarray scan images, extract .tif images using the e Extraction software.

n the Agilent Feature Extraction (FE) software.

et the most recent Feature Extraction protocols for gene expression, go he Agilent Web site at www.agilent.com/chem/feprotocols.

the images (.tif) to be extracted to the FE Project.

lick Add New Extraction Set(s) icon on the toolbar or right-click the roject Explorer and select Add Extraction...

rowse to the location of the .tif files, select the .tif file(s) and click pen. To select multiple files, use the Shift or Ctrl key when selecting.

FE program automatically assigns a default grid template and protocol each extraction set, if the following conditions are met:

or auto assignment of the grid template, the image must be generated rom an Agilent scanner and have an Agilent barcode.

or auto assignment of the Two-Color Gene Expression FE protocol, the efault Gene Expression protocol must be specified in the FE Grid emplate properties.

o access the FE Grid Template properties, double-click on the grid emplate in the Grid Template Browser.

FE Project Properties.

elect the Project Properties tab.

n the General section, enter your name in the Operator text box.

n the Input section, verify that at least the following default settings as hown in Figure 5 below are selected.

or FE 9.5, under Other, select GE2_QCM_Feb07. For FE 10.5 or higher, he metric sets are part of the protocol, so you do not need to set them.

2 Procedures Step 2. Extract data using Agilent Feature Extraction Software

46 Two-Color Micro

For outputs that can be imported into Rosetta Resolver, select MAGE and JPEG.

Figure

4 Che

a S

b Vte

ImGc

TTlwmarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

5 Default settings in FE 10.5.

ck the Extraction Set Configuration.

elect the Extraction Set Configuration tab.

erify that the correct grid template is assigned to each extraction set in he Grid Name column. To assign a different grid template to an xtraction set, select one from the pull down menu.

f a grid template is not available to select from the pull down menu, you ust add it to the Grid Template Browser. To add, right-click inside the rid Template Browser, select Add. Browse for the design file (.xml) and lick Open to load grid template into the FE database.

o update to the latest grid templates via Online Update, right-click Grid emplate Browser and select Online Update. You can also download the

atest grid templates from Agilent Web site at ww.agilent.com/chem/downloaddesignfiles. After downloading, you ust 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 p

c VPsmF

Td

IiBOa

des

6 Verihavextrpro

7 Sele

N These F

N Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 47

e the FE Project (.fep) by selecting File > Save As and browse for ired location.

fy that the icons for the image files in the FE Project Window no longer e a red X through them. A red X through the icon indicates that an action protocol was not selected. If needed, reselect the extraction

tocol for that image file.

ct Project > Start Extracting.5 Sav

OTEmany 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.

OTE If scans are done with an Agilent scanner in XDR mode, the High and Low images are automatically combined when imported into the Feature Extraction software version 9.1 or newer.rotocol to an extraction set.

erify that the correct protocol is assigned to each extraction set in the rotocol Name column. To assign a different protocol to an extraction et, select one from the pull down menu. For Agilent two-color icroarrays, select GE2-v5_95_Feb07 (in FE 9.5), GE2_105_Dec08 (in E 10.5) or GE2-107_Sep09 (in FE 10.7).

he protocols automatically distinguish the formats for processing the ata.

f a protocol is not available to select from the pull down menu, you must mport it to the FE Protocol Browser. To import, right-click FE Protocol rowser, select Import. Browse for the FE protocol (.xml) and click pen to load the protocol into the FE database. Visit the Agilent Web site t www.agilent.com/chem/feprotocols to download the latest protocols.

E Protocols were optimized using data from Agilent catalog arrays, which have


48 Two-Color Micro

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 insp

If a QCof the

Pro

QC valu

QC extr

Refer t(v8.1) 5989-3with thdownlwww.aarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

ecting Spot Finding at the Four Corners of the Array. See Figure 7.

Metric Set has been assigned to the FE Project, you can view the results metric evaluation in three ways:

ject Run Summary includes a summary sentence.

Report includes both a summary on the header and a table of metric es.

Chart includes a view of the values of each metric compared across all actions in FE Project.

o the application note on Use of Agilent Feature Extraction Software QC Report to Evaluate Microarray Performance (publication 056EN) for more details on quality assessment and troubleshooting e Feature Extraction QC Report. This technical note can be

oaded from the Agilent Web site at gilent.com/chem/dnaapplications.



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 eArrayserver

Figure Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 49

Login Setting dialog box, under Advanced Options, click Use eArray during extraction. See Figure 6.

6 eArray Login Setting


50 Two-Color Micro

Figure

QC Report - Agilent Technologies : 2 Color Gene Expression

Date Wednesday, November 25, 2009 - 10:17 BG Method No Background

Image

Protocol

User Nam

Grid

FE Versio

Sample(

Non U

Popularray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

7 Example of the first page of a QC Report for 4x44K microarray, generated by Feature Extraction Software

US23502353_251485044524_S01_H [1_2] Background Detrend On(FeatNCRange, LoPass)

GE2_107_Sep09 (Read Only) Multiplicative Detrend True

e Administrator Dye Norm Linear Lowess

014850_D_F_20090416 Linear DyeNorm Factor 1.87(Red) 4.07(Green)

n 10.7.1.1 Additive Error 6(Red)4(Green)

red/green) Saturation Value 642409 (r), 639098 (g)

Spot Finding of the Four Corners of the Array

Grid Normal

FeatureLocal Background

Red Green Red Green

niform 20 28 0 0

ation 108 105 840 762

Spatial Distribution of All Outliers on the Array532 rows x 85 columns

# FeatureNonUnif (Red or Green) = 32(0.07%)

# GeneNonUnif (Red or Green) = 25 (0.061 %)

BG NonUniform BG Population

Red FeaturePopulation Red Feature NonUniform

Green FeaturePopulationGreen Feature NonUniform

Net Signal Statistics

Agilent SpikeIns:

Red Green

# Saturated Features 0 0

99% of Sig. Distrib. 182642 24186

50% of Sig. Distrib. 24753 4055

1% of Sig. Distrib. 1211 190

Non-Control probes:

Red Green

# Saturated Features 0 0

99% of Sig. Distrib. 48156 24960



Red and Green Background Corrected Signals (Non-Control Inliers)

# Features (NonCtrl) with BGSubSignals < 0: 2837 (Red); 2376 (Green)


3Supplemental Procedures

The pr51Agilent Technologies

Absolute RNA Nanoprep Purification 52

Thermocycler Protocol 55

Quick Amp Labeling Kit Sample Preparation 58

Quality Assessment of Template RNA and Labeled cRNA 70

Preventing Ozone-Related Problems 76

ocedures in this chapter are optional but recommended.

3 Supplemental Procedures Absolute RNA Nanoprep Purification

52

Absolute RNA Nanoprep Purification

As an alternative to the Qiagen RNeasy purification columns, the Absolutely RNA NPreparwhen iRNA N

Step 1.

1 Pre

a I

1ll

b A8

5 mto 0

2 Pre

a A

b O(

c Tr

3 Pre

a A

c Tighten the cap on the container of High-Salt Wash Buffer and store at rTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocoloom temperature.b On the Low-Salt Wash Buffer container, mark the check box for 1x (Ethanol Added).anoprep kit can be used to purify the amplified cRNA after Step 2. e labeling reaction on page 23. Use the Absolutely RNA Nanoprep Kit t is necessary to concentrate purified cRNA samples. The Absolutely anoprep Kit uses an elution volume of 20 L.

Prepare the reagents

pare 80% sulfolane:

ncubate the 100% sulfolane in a 37C water bath until liquefied.

00% sulfolane is a solid at room temperature. 80% sulfolane solution is a iquid at room temperature and can be stored at room temperature for at east a month.

dd 1 mL of RNase-free water to 4 mL of 100% sulfolane to make 5 mL of 0% sulfolane.

L of 80% sulfolane is enough to process 50 RNA preparations (from up .1 mL lysate each).

pare 1x high-salt wash buffer:

dd 16 mL of 100% ethanol to the bottle of 1.67X High-Salt Wash Buffer.

n the High-Salt Wash Buffer container, mark the check box for 1x Ethanol Added).

ighten the cap on the container of High-Salt Wash Buffer and store at oom temperature.

pare the 1x low-salt wash buffer:

dd 68 mL of 100% ethanol to the bottle of 5X Low-Salt Wash Buffer.

Supplemental Procedures 3Step 2. Purify the labeled/amplified RNA

Two-Co


1 Add 100 L of the Lysis Buffer to each reaction tube for a total volume of 116

2 Mix

3 Addcell

Youvort

4 Put

5 Tranan

6 Spin

7 Reminto

Up

9 Reminto

10 Addand

11 Rep

12 Reminto

13 Addandthe

14 Tra

CAUTIONlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 53ove and keep the spin cup. Discard the filtrate. Put the spin cup back the same 2-mL collection tube.

300 L of 1x Low-Salt Wash Buffer to the spin cup. Cap the spin cup, spin the sample in a microcentrifuge at 12,000 x g for 60 seconds.eat step 9 and step 10 for a second low-salt wash.

ove and keep the spin cup. Discard the filtrate. Put the spin cup back the same 2-mL collection tube.

300 L of 1x Low-Salt Wash Buffer to the spin cup. Cap the spin cup, spin the sample in a microcentrifuge at 12,000 x g for 3 minutes to dry fiber matrix.

nsfer the spin cup to a fresh 2-mL collection tube.

h-Salt Wash Buffer contains the irritant guanidine thiocyanate.of guanidine thiocyanate.

8 Add 300 L of 1x High-Salt Wash Buffer to the spin cup. Cap the spin cup, and spin the sample in a microcentrifuge at 12,000 x g for 60 seconds.

The Hig L.

on a vortex mixer, or pipette repeatedly until homogenized.

an equal volume (116 L) of 80% sulfolane (room temperature) to the lysate. Mix thoroughly on a vortex mixer for 5 seconds.

must use equal volumes of 80% sulfolane and cRNA sample. Mix on a ex mixer until the cRNA sample and sulfolane are thoroughly mixed.

an RNA-binding nano-spin cup into a 2-mL collection tube.

nsfer the 80% sulfolane and cRNA sample mixture to the RNA-binding o-spin cup and snap the cap onto the top of the spin cup.

the sample in a microcentrifuge at 12,000 x g for 60 seconds.ove and keep the spin cup. Discard the filtrate. Put the spin cup back the same 2-mL collection tube.

to this point, the RNA has been protected from RNases by the presence

3 Supplemental Procedures Step 2. Purify the labeled/amplified RNA

54 Two-Color Micro

15 Add 20 L of Elution Buffer directly onto the fiber matrix inside the spin cup. Cap the spin cup and incubate the sample at room temperature for 2 minutes.

To i

16 Spin

17 If nof t

18 Trato s

Thelong

NOTE The Elupermeaarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolncrease the RNA yield, warm the Elution Buffer to 60C.

the sample in a microcentrifuge at 12,000 x g for 5 minutes.eeded, repeat the elution step (step 15 and step 16) to increase the yield otal RNA.

nsfer the eluate in the collection tube to a capped microcentrifuge tube tore the RNA.

RNA can be stored at -20C for up to one month, or at 80C for -term storage.

tion Buffer must be added directly onto the fiber matrix so that the buffer can te the entire fiber matrix.

Supplemental Procedures 3Thermocycler Protocol

Two-Co

Thermocycler Protocol

The procedure in this section is an optional thermocycler protocol for the Low

Nlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 55Input Quick Amp Labeling Kit.

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: 65C for 10 minutes, 4C hold

Program 2: 40C for 2 hours, 70C for 15 minutes, 4C hold

Program 3: 40C for 2 hours, 4C hold

Five minutes at 4C is enough. Hold at that temperature if the reagents for the next step are not ready.

OTE Use a heated lid for optimal results.

3 Supplemental Procedures Step 2. Synthesize cDNA from Total RNA

56

Step 2. Synthesize cDNA from Total RNA

(Time required: ~3 hours)

3 ImmTab

4 Addtota

8 To e10

9 Putdou

NOTE Prewarminutesresuspedrive th

NOTE Keep thMix unt

NOTE IncubatTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolach sample tube, add 4.7 L of cDNA Master Mix for a total volume of L. Pipette up and down to mix.

reaction tubes in thermocycler and run Program 2 to synthesize ble-stranded cDNA.

m the 5X first strand buffer by incubating the vial in an 80C water bath for 3 to 4 to ensure adequate resuspension of the buffer components. For optimal nsion, mix briefly on a vortex mixer and spin the tube briefly in a microcentrifuge to e contents off the tube walls. Keep at room temperature until use.

e AffinityScript RNase Block Mix on ice. Do not add the AffinityScript RNase Block il just before you start the reactions.

ion at 70C inactivates the AffinityScript enzyme.total volume of 5.3 L.

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 4C, or move to benchtop rack on ice.

7 Immediately prior to use, gently mix the components in Table 3 on page 25 in the order listed by pipetting, and keep at room temperature.ediately prior to use, use a pipette to gently mix the components in le 2 on page 24.

1.8 L of T7 Promoter Primer Mix to the tube that contains 3.5 L of l RNA and diluted RNA spike-in controls. Each tube now contains a 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 L. For optimal performance, use at least 25 ng of input total RNA.

2 Add 2 L of the diluted Two-Color Spike Mix. Please refer to Table 1 on page 20 for detailed instructions on the preparation and use of Spike-in kits.

Supplemental Procedures 3Step 3. Synthesize Fluorescent cRNA Synthesis in vitro

Two-Co

Step 3. Synthesize Fluorescent cRNA Synthesis in vitro


1 Imm

a As

b M

c A

d Mb

2 Keemov

3 To epipe

4 RetPro

5 Purlabe

NOTE Do not do the rlor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 57ediately before use, make Master Mix for each cyanine dye:

dd the first four components listed in Table 4 on page 26 in the order hown to 1.5 mL nuclease-free microfuge tubes at room temperature.

ix thoroughly on a vortex mixer.

dd the T7 RNA Polymerase Blend and cyanine dyes.

ix gently, but completely, by pipetting up and down without introducing ubbles.

p the reaction tubes from step 9 above in the thermocycler at 4C, or e to benchtop rack on ice.

ach sample tube, add 6 L of Transcription Master Mix. Gently mix by tting up and down. The final volume of the reaction is now 16 L.

urn the reaction tubes to the thermocycler and run Program 3 (Step 1. gram the thermocycler on page 55) to synthesize labeled cRNA.

ify the labeled cRNA as described on Step 3. Purify the led/amplified RNA on page 27.

add the T7 RNA Polymerase Blend to Transcription Master Mix until just before you eaction.

3 Supplemental Procedures Quick Amp Labeling Kit Sample Preparation

58

Quick Amp Labeling Kit Sample Preparation

The Low Input Quick Amp Labeling Kit replaces the Quick Amp Labeling Kit. If you hapreparInput

AgilenRNA) wminimT7 RNincorp100-fo

NOTE For optirefer toguidancTwo-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolve studies that are ongoing, continue to use the Quick Amp Labeling ation steps described in this section. For new studies, use the Low

Quick Amp Labeling Kit as described in Chapter 2, Procedures.

t's Quick Amp Labeling Kit generates fluorescent cRNA (complimentary ith a sample input RNA range between 50 ng and 5 g of total RNA or a

um of 10 ng of poly A+ RNA for two-color processing. The method uses A polymerase, which simultaneously amplifies target material and orates cyanine 3- or cyanine 5-labeled CTP. There is routinely at least a ld RNA amplification with use of this kit.

mal performance, use high quality, intact template total or poly A+ RNA. Please Quality Assessment of Template RNA and Labeled cRNA on page 70 for general e and procedural recommendations on quality assessment of template RNA.

Supplemental Procedures 3Quick Amp Labeling Kit Sample Preparation


Figure Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 59

8 Schematic of amplified cRNA procedure. Generation of cRNA for a two-color microarray experiment is shown. When you generate targets for a one-color microarray experiment, only the Cy3-labeled B sample is produced and hybridized.

3 Supplemental Procedures Step 1. Prepare Spike A Mix and Spike B Mix

60

Step 1. Prepare Spike A Mix and Spike B Mix

Refer to the protocol on Agilent RNA Spike-In Kit (publication 5188-5928) for in-depspike-ican alswww.a

1 Equ

2 Mix

3 Hea

4 Brieopeoccu

Table differeAlwayoutputgeneraorientthe po

Table 1

Starting

Total RN(ng)

50-200

201-200

>2000Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolth instructions and troubleshooting advice on how to use two-color ns. This protocol is available with the Two-Color RNA Spike-In Kit and o be downloaded from the Agilent Web site at gilent.com/chem/dnamanuals-protocols.

ilibrate water baths to 37C, 65C, 40C, and 80C.

the stock solutions vigorously on a vortex mixer.

t at 37C for 5 minutes, and mix on a vortex mixer once more.

fly centrifuge to drive contents to the bottom of the tube prior to ning. Settlement of the solution on the sides or lid of the tubes may r during shipment and storage.

13 provides the dilutions of Spike A Mix and Spike B Mix for three nt starting sample ranges of total RNA or 0.2 g of poly A mRNA. s label Spike A Mix with cyanine 3 and Spike B Mix with cyanine 5. The of the Agilent SpikeIns LogRatio Statistics table in the QC report, ted when using Agilent Feature Extraction Software 9.5.3, uses this

ation. It is built into the software code and cannot be set by the user. If larity is flipped, you will have to manually adjust the output.

3 Dilutions of Spike A Mix for cyanine 3 and Spike B Mix for cyanine 5 labeling

amount of RNA Serial dilution Spike A Mix or Spike B Mix volume to be used (L)

A PolyARNA (ng)

First Second Third

1:20 1:40 1:16 2

0 1:20 1:40 1:4 2

200 1:20 1:40 0 2

Supplemental Procedures 3Step 1. Prepare Spike A Mix and Spike B Mix

Two-Co

For extotal R

1 Mak

a M

b H

c M

a LD

b I

c A

d Mtt

2 Mak

a LD

b I

c A

d Mtt

3 Mak

a LD

b I

c A

d Mtt

NOTE Use RNase-free microfuge tubes and tips. Avoid pipetting volumes less than 2 L to ensure accuracy.lor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 61ample, to prepare the Spike A Mix dilution appropriate for 200 ng of NA starting sample:

e the First Dilution:

ix the thawed Spike Mix vigorously on a vortex mixer.

eat at 37C in a circulating water bath for 5 minutes.

ix the Spike Mix tube vigorously again on a vortex mixer.

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix First ilution.

nto the First Dilution tube, put 2 L of the concentrated Spike A Mix.

dd 38 L of the Dilution Buffer, provided in the Spike-In kit (1:20).

ix thoroughly on a vortex mixer and spin down quickly in a centrifuge o collect all of the liquid at the bottom of the tube. This tube contains he First Dilution.

e the Second Dilution:

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix Second ilution.

nto the Second Dilution tube, put 2 L from the First Dilution tube.

dd 78 L of the Dilution Buffer (1:40).

ix thoroughly on a vortex mixer and spin down quickly in a centrifuge o collect all of the liquid at the bottom of the tube. This tube contains he Second Dilution.

e the Third Dilution:

abel a new sterile 1.5 mL microcentrifuge tube Spike A Mix Third ilution.

nto the Third Dilution tube, put 2 L from the Second Dilution tube.

dd 30 L of the Dilution Buffer (1:16).

ix thoroughly on a vortex mixer and spin down quickly in a centrifuge o collect all of the liquid at the bottom of the tube. This tube contains he Third Dilution.

3 Supplemental Procedures Step 1. Prepare Spike A Mix and Spike B Mix

62 Two-Color Micro

Storage of Spike Mix dilutions

Store the Agilent RNA Spike-In Kit, Two-Color at 70C to 80C in a non-defrosting freezer for up to 1 year from the date of receipt.

The firstoredfreeze/

After uarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

st dilution of the Agilent Spike Mix (A or B) positive controls can be up to 2 months in a non-defrosting freezer at 70C to 80C and thawed up to eight times.

se, discard the second and third dilution tubes.

Supplemental Procedures 3Step 2. Prepare labeling reaction

Two-Co

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 aof s

2 Pre5-C

3 AddTwo

4 Add

5 Use

6 Dena ci

7 Plac

8 Immthe tem

9 Preaderesumicroo

MMto b

Be s

Table 1

RNA vol

8.3lor Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 63ppropriate volume of 8.3 L or less. Dilute samples so that at least 2 L ample is pipetted into the tube.

pare tubes for both Spike A Mix/cyanine 3-CTP and Spike B Mix/cyanine TP dyes.

1.2 L of T7 Promoter Primer (from the Agilent Quick Amp Kit, -Color). See Table 14.

2 L of diluted Spike Mix (A or B).

nuclease-free water to bring the total reaction volume to 11.5 L.

ature the primer and the template by incubating the reaction at 65C in rculating water bath for 10 minutes.

e the reactions on ice and incubate for 5 minutes.

ediately prior to use, gently mix the components listed in Table 15 for cDNA Master Mix by adding in the order indicated, and keep at room perature.

warm the 5X first strand buffer at 80C for 3 to 4 minutes to ensure quate resuspensions of the buffer components. For optimal spension, briefly mix on a vortex mixer and spin the tube in a

rocentrifuge to drive down the contents from the tube walls. Keep at m temperature until needed.

LV-RT and RNaseOUT are enzymes, which need to be kept on ice and are e added to the cDNA Master Mix just before starting the reactions.

ure to use the 10 mM dNTP mix tube from the kit.

4 Template (RNA Spike A or B Mix) and T7 Promoter Primer Mix

ume (L) Diluted Spike A (or B) Mix amounts (L)

T7 Promoter primer (L)

Total volume (L)

2 1.2 11.5

3 Supplemental Procedures Step 2. Prepare labeling reaction

64 Two-Color Micro

10 Briecon

11 Addup a

12 Incu

13 Mov

14 Mov

15 Spinthe

16 Immthe tem

17 Preresumicpipeunt

RNaenzTra

Table 15 cDNA Master Mix

Component Volume (L) per Volume (L) per 4.5

5X First

0.1 M DT

10 mM d

MMLV-R

RNaseO

Total Voarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol

fly spin each sample tube in a microcentrifuge to drive down the tents from the tube walls and the lid.

8.5 L of cDNA Master Mix to each sample tube and mix by pipetting nd down.

bate samples at 40C in a circulating water bath for 2 hours.

e samples to a 65C circulating water bath and incubate for 15 minutes.

e samples to ice. Incubate for 5 minutes.

samples briefly in a microcentrifuge to drive down tube contents from tube walls and lid.

ediately prior to use, gently mix the components listed in Table 16 in order indicated for the Transcription Master Mix by pipetting at room perature.

warm the 50% PEG solution at 40C for 1 minute. For optimal spension, briefly mix on a vortex mixer and spin the tube in a

rocentrifuge to drive down the contents from the tube walls. Careful tting is required to ensure accurate volume. Keep at room temperature

il needed.

seOUT, inorganic pyrophosphatase, and T7 RNA polymerase are ymes, which need to be kept on ice and should be added to the nscription Master Mix just before starting the reactions.

reaction reactions

Strand Buffer 4 18

T 2 9

NTP mix 1 4.5

T 1 4.5

ut 0.5 2.3

lume 8.5 38.3

Supplemental Procedures 3Step 2. Prepare labeling reaction


18 Addpipe

19 Incu

Table 16 Transcription Master Mix

Component Volume (L) per Volume (L) per 4.5

Nucleas

4X Trans

0.1 M DT

NTP mix

50% PEG

RNaseO

Inorgani

T7 RNA

Cyanine

Total Vo Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 65

60 L of Transcription Master Mix to each sample tube. Gently mix by tting.

bate samples in a circulating water bath at 40C for 2 hours.

reaction reactions

e-free water 15.3 68.9

cription Buffer 20 90

T 6 27

8 36

6.4 28.8

UT 0.5 2.3

c pyrophosphatase 0.6 2.7

Polymerase 0.8 3.6

3-CTP or cyanine 5-CTP 2.4 10.8

lume 60 270

3 Supplemental Procedures Step 3. Purify the labeled/amplified RNA

66


Qiagens RNeasy mini spin columns are recommended for purification of the

2 m13,0

5 Trabuff4Ccoll

6 Add4Ccoll

7 If anRNe4CRPEcRN

N

CAUTION Do not Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocolnsfer the 700 L of the cRNA sample to an RNeasy mini column in a L collection tube. Centrifuge the sample at 4C for 30 seconds at 00 rpm. Discard the flow-through and collection tube.

nsfer the RNeasy column to a new collection tube and add 500 L of er RPE (containing ethanol) to the column. Centrifuge the sample at for 30 seconds at 13,000 rpm. Discard the flow-through. Re-use the ection tube.

another 500 L of buffer RPE to the column. Centrifuge the sample at for 60 seconds at 13,000 rpm. Discard the flow-through and the ection tube.

y buffer RPE remains on or near the frit of the column, transfer the asy column to a new 1.5 mL collection tube and centrifuge the sample at for 30 seconds at 13,000 rpm to remove any remaining traces of buffer . Discard this collection tube and use a fresh tube to elute the cleaned A sample.

discard the final flow-through in the next step. It contains the cRNA sample.amplified cRNA samples.

1 Add 20 L of nuclease-free water to your cRNA sample, for a t

agilent geneexpression two color v6.5

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