Enzo Life Sciences Data Sheet

Cyanine Labeling for Gene Expression Analysis

Enzo Solves the Problems ofCyanine Labeling

All parameters of the GeneBeam™

system have been optimized toassure maximum performance ofmicroarrays. Enzo has carefullyadjusted the critical cyanine dye-dUTP to cognate nucleotide ratio toassure the perfect balance betweenmaximized yield and maximizedlabeling efficiency. This critical anddistinguishing feature of theGeneBeam™ system alleviates theneed for lengthy multistep indirectlabeling methods. The GeneBeam™

protocol yields standardized andreproducible gene expression data.

Enzo’s unique dye-labeled nucleotides,cyanine-5-dUTP and cyanine-3-dUTP, are prepared using a novellinker arm for dye attachment. Thislinkage assures efficient incorpora-tion of dye molecules into cDNA.The buffer and enzyme formulationshave been carefully optimized toassure excellent performance.

Another outstanding benefit of theGeneBeam™ system is that reac-tions have been optimized so thefluorescence intensities of bothdyes are nearly equivalent whenequal masses of total RNA startingmaterial are labeled in the cyanine-

3 and cyanine-5 labeling reactions.In typical cyanine dye labeling reac-tions, the incorporation of cy3-labeled nucleotides is higher thanthe incorporation of cy5-nucleotides. However, the quantumyield (a measure of the degree offluorescence) of cy3 is much lowerthan that of cy5. Enzo’s highly opti-mized GeneBeam™ labeling sys-tem procedure yields cyanine-3 andcyanine-5-labeled cDNA prepara-tions in which intensity valuesrequire minimal normalization dur-ing data processing. This featureclearly demonstrates why indirectlabeling methods are no longer nec-essary.

GeneBeam™ First Strand cDNA Labeling System

Applications: + cDNA and Oligonucleotide Microarrays for Gene Expression Analysis

Advantages: + Eliminates need for lengthy indirect labeling protocol with a 2.5 hour procedure.

+ Maximizes yield and labeling efficiency with fully optimized nucleotide ratios.

+ Minimizes normalization of intensity values during data processing.

+ Saves time and money by offering all components, including cyanine-labeled nucleotides in one system.

+ Provides low background, high signal and high sen-sitivity in microarray experiments.

+ Decreases inherent labeling bias since fluorescence intensities of both dyes are nearly equivalent.

+ Simplifies traditional indirect protocol with fewer purifications.

+ Performs well with a broad range of slide surface chemistries.

Microarray Analysis

Figure 1: Direct vs. Indirect Labeling

42530v01 04/01/2005

The GeneBeam™ First StrandcDNA Labeling System containsreagents for the production of fluo-rescently labeled cDNA for use indual-color microarray assays. Thisfully optimized system is supplied ina straightforward, ready-to-use for-mat with a rapid procedure totaling2.5 hours. The GeneBeam™ sys-tem solves the current problems ofcyanine labeling and eliminates theneed for indirect labeling by provid-ing even incorporation of dyes, lowbackground and high signal intensi-ties.

A. GeneBeam™ direct labeling B. Indirect labeling

GeneBeam™ First Strand cDNA Labeling System

Catalog Number Description Quantity

42530 GeneBeam™ First Strand cDNA LabelingSystem

2 x 25 reactions

42501 Cyanine-3-dUTP 25 nmol

42502 Cyanine-5-dUTP 25 nmol

Patents:This product and the use of this product is covered by one or more claims of Enzo patents, including but not lim-ited to the following: U.S. Patent Nos. 4,994,373, 5,175,269; Canadian Patent Nos. 1,309,672, 1,314,503,1,341,365, and patents pending.

Enzo, BioArray, GeneBeam and ENZ-RT are trademarks of Enzo Life Sciences, Inc. All other products and serv-ices mentioned are trademarks or service marks of their respective companies or organizations.

Research Use Only:This product is sold by Enzo Life Sciences, Inc. for research purposes only by the end user in the research marketand is not intended for diagnostic or therapeutic use. Purchase does not include or carry any right or license to use,develop or otherwise exploit this product commercially. Any commercial use, development or exploitation of this productor development using this product without the express prior written authorization of Enzo Life Sciences, Inc. is strictlyprohibited.

The GeneBeam™ First-Strand cDNA Labeling System contains Enzo's cyanine-3-dUTP and cyanine-5-dUTP.These proprietary nucleotides are prepared utilizing a novel allylic reactive group for direct attachment of the dyeto a mercurated nucleotide through a novel semi-rigid linker arm.

Copyright ©2005 Enzo Life Sciences, Inc. All rights reserved.

Ordering Information >>

Product Components >>

For more information or to place an order, contact your Enzo Life Sciences’ Alliance Manager at1.800.221.7705 or 1.631.694.7070 or visit our web site at www.enzolifesciences.com.

The GeneBeam™ First Strand cDNA Labeling System contains all necessary reagents for theproduction of fluorescently labeled cDNA for use in dual-color microarray assays.

Reagent Description Quantity

Oligo dT Primer poly dT in Tris buffer 55 µL

5X Reaction Buffer Tris buffer with enhancers 250 µL

10X DTT 100 mM in aqueous solution 110 µL

Cyanine 3-dUTPDeoxynucleotide Mix

Cy-3-dUTP with dTTP, dATP, dCTP and dGTP 55 µL

Cyanine 5-dUTPDeoxynucleotide Mix

Cy-5-dUTP with dTTP, dATP, dCTP and dGTP 55 µL

ENZ-RT™ / RNaseInhibitor

Reverse Transcriptase and RNase mix instorage buffer

110 µL

RNase Cocktail RNase in storage buffer 110 µL

Control RNA 1 µg/µL of mRNA 8 µL

Nuclease-free Water 1 mL

Enzo Life Sciences, Inc.

60 Executive Boulevard

Farmingdale, NY 11735

Toll Free: 1.800.221.7705

International: 1.631.694.7070

Fax: 1.631.694.7501

enzolifesciences@enzobio.com

www.enzolifesciences.com

GeneBeam ™ First-Strand cDNA Labeling Kit

for preparation of Cyanine-3- and Cyanine-5-labeled cDNA for Microarray Hybridization

An Optimized, Ready-to-Use BioArray ™ Product

INSTRUCTION MANUAL product number

42530 50 labeling reactions (25 reactions for labeling with cyanine-3-dUTP and 25 reactions for labeling with cyanine-5-dUTP)

For research use only.

5

Version 4.3.0, June 200

Technical Support Toll free from the U.S. and Canada: 1-800-221-7705 All others: 631-694-7070 Fax: 631-694-7501 email: enzolifesciences@enzobio.com http://www.enzolifesciences.com

Notice to Purchaser This product is sold by ENZO LIFE SCIENCES, INC. for research purposes only by the end-user in the research market and is not intended for diagnostic or therapeutic use. Purchase does not include any right or license to use, develop or otherwise exploit this product commercially. Any commercial use, development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES, INC. is strictly prohibited.

Limited Warranty These products are offered under a limited warranty. The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment. Enzo Life Sciences’ sole obligation is to replace the product to the extent of the purchase price. All claims must be made to Enzo Life Sciences, Inc. within five (5) days of receipt of order.

Patents This product and the use of this product is covered by one or more claims of Enzo patents, including but not limited to the following: U.S. Patent Nos. 5,328,824; 5,449,767; 5,476,928; 4,994,373 and 5,175,269; Canadian Patent Nos. 1,309,672; 1,314,503 and 1,341,365; and patents pending.

Trademarks BioArray, GeneBeam and ENZ-RT are trademarks of Enzo Life Sciences, Inc. QIAquick is a registered trademark of Qiagen. SYBR is a registered trademark of Molecular Probes, Inc. Typhoon is a trademark of Amersham Pharmacia Biotech. Lifter Slips is a trademark of Erie Scientific

Additional Note The GeneBeam ™ First-Strand cDNA Labeling Kit contains Enzo’s cyanine-3-dUTP and cyanine-5-dUTP. These proprietary nucleotides are prepared utilizing a novel allylic reactive group for direct attachment of the dye to a mercurated nucleotide through a novel semi-rigid linker arm.

GeneBeam™ First-Strand cDNA Labeling Kit

CONTENTS

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

Description........................................................................ 1

System outline.................................................................. 2

Components ..................................................................... 3

Storage conditions........................................................... 3

Safety warnings and precautions................................... 4

Additional materials required ......................................... 4

Procedures................................................................... 5-14

A. RNA Analysis........................................................ 5

B. Labeling first-strand cDNA with Cyanine-3- or Cyanine-5-nucleotides .................................... 5

C. Purification of Labeled cDNAs.............................. 9

D. Determination of Incorporation of Cyanine-Labeled Nucleotide and Yield .............. 10

E. Size Distribution Analysis By Gel Electrophoresis................................................... 12

F. Preparation of Labeled cDNA for Microarray Hybridization..................................... 12

G. Procedure Validation .......................................... 13

Recommendations for hybridization............................ 14

Troubleshooting guide .................................................. 15

References ...................................................................... 16

1

INTRODUCTION Nucleic acid microarray analysis can simultaneously determine the relative abundance of a large number of mRNA species. This can be accomplished by reverse transcriptase-catalyzed conversion of an mRNA population to a fluorescently-labeled cDNA target population. This population is then probed by hybridization to a large set of complementary nucleic acid sequences (probes) arrayed onto the surface of a glass slide. The intensity of the fluorescent signal emanating from each spot is directly proportional to the number of copies of a specific mRNA species in the labeled population. By labeling reference and experimental mRNA populations with fluorescent dyes that have distinct emission wavelengths, the relative expression levels of large numbers of genes can be determined quickly and reliably on a single array.

Enzo’s GeneBeam™ First-Strand cDNA Labeling Kit has been developed to provide reliable and reproducible production of fluorescently labeled cDNA. This system utilizes Enzo’s proprietary cyanine dye-labeled nucleotides. These nucleotides are prepared utilizing a novel allylic reactive group for direct attachment of the dye to a mercurated nucleotide through a novel semi-rigid linker arm. Dye-labeled nucleotides prepared by this proprietary method are efficiently incorporated into cDNA by Enzo’s ENZ-RT ™ reverse transcriptase. The resulting labeled cDNA generates strong signals with low backgrounds on a variety of microarray substrates.

The GeneBeam ™ First-Strand cDNA Labeling Kit is supplied

in a complete, ready-to-use format and is fully optimized to produce cDNAs with maximum incorporation of cyanine dye-labeled nucleotides. Excellent results can be obtained from total RNA samples as small as 10 µg and purified mRNA samples as low as 100 ng.

DESCRIPTION

The GeneBeam ™ First-Strand cDNA Labeling Kit contains

reagents for the production of fluorescently labeled cDNA for use in dual-color microarray assays. This fully optimized system is supplied in a straightforward, ready-to-use format. The kit also includes a control RNA that can be used as a template for cDNA synthesis in control reactions.

SYSTEM

The procedure begins with the annealing of oligo dT primers to the poly A sequence of an mRNA. Labeled cDNA is generated by primer extension catalyzed by ENZ-RT

™ reversetranscriptase in the presence of a nucleotide mix containing either cyanine-3-dUTP or cyanine-5-dUTP. The RNA template is then degraded and the labeled cDNA is purified prior to hybridization in microarray assays.

OUTLINE The scheme below outlines the steps of the system:

Primer Hybridization

Reverse Transcription

Template Degradation + RNase

+ ENZ-RT ™ + Cyanine-5-dUTP

+ ENZ-RT ™ + Cyanine-3-dUTP

Denature + Oligo dT

5’ AAAAAA 3’ T T T T T T 5’

Denature + Oligo dT

Starting Reference RNA

5’ AAAAAA 3’

AAAAAA 3’ 5’3’ T T T T T T 5’

+ RNase

3’ T T T T T T 5’ 3’ T T T T T T 5’

Combined Purified Labeled cDNAs

LEGEND: RNA DNA Cyanine-3 Cyanine-5

Sample Array

Column Purification

AAAAAA AAA AAA

AAAAAA 3’ 5’3’ T T T T T T 5’

5’ AAAAAA 3’ T T T T T T 5’

5’ AAAAAA 3’

Starting Experimental RNA

2

3

COMPONENTS

42530a

Oligo dT Primer (dT) 55 µL 5X Reaction Buffer (B) 250 µL 10X DTT (D) 110 µL Cyanine-3-dUTP Deoxynucleotide Mix (green)

55 µL

Cyanine-5-dUTP Deoxynucleotide Mix (red)

55 µL

ENZ-RT ™ / RNase Inhibitor (RT) 110 µL

RNase Cocktail (yellow) 110 µL

Control RNA, 1 µg/µL (C) 8 µL Nuclease-free water (W) 1 mL

aSufficient reagents and materials are provided for 50 reactions.

STORAGE CONDITIONS

The GeneBeam ™ First-Strand cDNA Labeling Kit is shipped

on dry ice. Upon receipt, store all reagents at –20°C, in a non-frost free freezer. Avoid repeated freezing and thawing of the cyanine-labeled deoxynucleotides and the enzymes. For long-term storage, store the reagents at –80°C.

Cyanine-3-dUTP and Cyanine-5-dUTP are light sensitive. Protect from light exposure at all times.

The product is stable until the expiration date indicated on the box when stored properly.

SAFETY WARNINGS AND PRECAUTIONS • This product is for research use only. It is not intended for

diagnosis of diseases in humans or animals. DO NOT use internally or externally in humans or animals.

• CyaninPerform all reactions in amber microcentrifuge tubes or protected from light by other means. Take precautions to prevent RNA deg

e-3-dUTP and Cyanine-5-dUTP are light sensitive.

• radation. Wear isposable powder-free laboratory gloves during the entire

t

• also ns DTT (dithiothreitol), can cause irritation to the skin,

is harmful if swallowed or

ADDITIONAL The following materials are required but not provided with the

beling Kit. All reagents d free of any

terile, a ber microcentrifuge tubes (nuclease-free

aterbath, PCR Machine, oven or heating block set to 70°C 42°C

set to 37°C e bath

• Hybridization chambers

dprocedure and use nuclease-free aerosol-resistant pipetips. The 10X DTT (D) and ENZ-RT

™/RNase Inhibitor (RT), whichcontaieyes and respiratory tract. It inhaled. Should these solutions come in contact with skin or eyes, wash immediately with water.

MATERIALS REQUIRED

GeneBeam ™ First-Strand cDNA La

should be molecular biology grade ancontaminating nucleases. • QIAquick® PCR Purification Kit (Qiagen No. 28104) • Micropipets, 1 µL to 1mL • S m ) • Sterile, nuclease-free aerosol barrier pipet tips • Microcentrifuge, tabletop • W• Waterbath, PCR Machine, oven or heating block set to• Waterbath, PCR Machine, oven or heating block• Ic• Lyophilizer • Lifter Slips

™ (Erie Scientific)

4

5

PROCEDUASE READ THE ENTIRE PROCEDURE BEFORE STARTING.

A. RNA ANALYSIS d in the labeling

itical for successful array analysis. e gel analysis should be

ples prior to labeling and concentrated RNA solution should

r, and the A260/A280 ratio should be above 1.8. If this is

To assure the integrity of the starting material analyze 1µg to ple in a 1-2% agarose gel visualized using

ot er and 8S) ared

.

ier.

B.

The primer is annealed to the RNA in a mix of water, primer ing

. If several arrays are to ipeting errors can be

reduced by assembling sufficient volumes of the reference

RES PLE

The purity and integrity of the RNA usereactions is crSpectrophotometric and agarosperformed on the RNA sam

ybrid zation. The purified, h ibe cleanot the case, do not continue with the cDNA synthesis.

2µg of the RNA samSYBR™ Gold or other dye. A denaturing gel system is nnecessary if the samples are loaded in a denaturing buffheated prior to gel loading. Discreet bands seen at 5kb (2and 1.9kb (18S) indicate intact RNA, while diffuse or smebands suggest degradation. If degradation is observed a fresh RNA sample should be prepared. Transfer RNA (tRNA) and 5SRNA may sometimes appear as diffuse and faster-migrating bands. DNA contamination can appear as distinct high molecular weight bands that migrate slower than the 28S bandDNA contamination can be eliminated by treatment with RNase-free DNase I according to instructions provided by the suppl

LABELING FIRST-STRAND CDNA WITH CYANINE-3- AND CYANINE-5-NUCLEOTIDES

NOTE Before beginning, prepare water baths or heating blocks at

70°C, 42°C and 37°C, and an ice bath.

DENATURE TARGET RNA AND ANNEAL PRIMERS

and the RNA sample. The volumes indicated in the followsteps are for one annealing reactionbe run using the same reference RNA, p

RNA, w l and distributing into ure to include a 10% overage of each component when assembling

)

e

sary, and then incubate at 70°C for 10 minutes to

4. Keep on

Two array

indi

ted below by the number of arrays being performed (see Table 1 on page 7). The volumes indicated in Table de a 10% overage to

ipeting.

ater and primer (see Table 1 on page 7) as a poo separate labeling reactions. Be s

the pool. +1. Add total RNA (10-50 µg) or poly A RNA (100 ng to1 µg

to a sterile nuclease-free reaction tube. If the concentration of the experimental RNA is low, lyophilize the appropriate volume and raise to 9 µL with Nuclease-free Water (W).

2. Add 1 µL of Oligo dT Primer (dT) and bring the total samplvolume to 10 µL with Nuclease-free Water (W).

3. Mix gently by flicking the tube a few times, spin down if necesdenature secondary structure in the RNA.

Cool the reaction mixture on ice for 2 minutes. ice until ready for the next step.

PREPARE REACTION MASTER MIX

labeling reactions must be prepared for each microassay: one for labeling with Cyanine-3-dUTP and one for labeling with Cyanine-5-dUTP. The reagent volumes

cated in the following example are for one array.

5. For each array to be run, prepare a Master Mix sufficient for two labeling reactions. When running more than one array multiply the reagent volumes indica

1 inclucompensate for losses due to p

Component Vol / Array 5X Reaction Buffer (B) 8 µL 10X DTT (D) 4 µL ENZ-RT

™/RNase Inhibitor (RT) 4 µL Total 16 µL

Keep the Master Mix on ice and continue.

6

10

11.0

110.0

10

20

88

44.0

44.0

88.0

22.0

22.0

9 9.9

99.0 9 18

79.2

39.6

39.6

79.2

19.8

19.8

0 4 2 2 4 6 6

Tabl

e 1.

Vol

ume

Req

uire

men

ts fo

r Mul

tiple

Rea

ctio

ns

7

8 8.8 88. 8 16 70.

35.

35.

70.

17.

17.

7 7.7

77.0 7 14

61.6

30.8

30.8

61.6

15.4

15.4

6 6.6

66.0 6 12

52.8

26.4

26.4

52.8

13.2

13.2

5 5.5

55.0 5 10

44.0

22.0

22.0

44.0

11.0

11.0

4 4.4

44.0 4 8 35.2

17.6

17.6

35.2 8.8

8.8

3 3.3

33.0 3 6 26.4

13.2

13.2

26.4 6.6

6.6

2 2.2

22.0 2 4 17..6

8.8

8.8

17.6 4.4

4.4

1 2 8.8

4.4

4.4

8.8

2.2

2.2

A 10

% o

vera

ge is

inclu

ded

in th

e fol

lowi

ng to

ensu

re su

fficie

nt vo

lum

e.

No. o

f Rea

ction

s

Oligo

dT P

rimer

(dT)

RNA

+ Prim

er +

Nuc

lease

-free

Wate

r (W

)

No. o

f Arra

ys

No. o

f Rea

ction

s

5X R

eacti

on B

uffer

(B)

10X

DTT

(D)

ENZ-

RT™

/ RNa

se in

hibito

r (RT

)

Pipe

t the

indi

cate

d vo

lum

e int

o tw

o ali

quot

s (A

and

B)

Cyan

ine-3

-dUT

P D

eoxu

nucle

otide

Mix

into a

liquo

t A a

Cyan

ine-5

-dUT

P D

eoxu

nucle

otide

Mix

into a

liquo

t B a

a Use

10

µL a

liquo

t A fo

r Exp

erim

enta

l RNA

labe

ling

and

10 µ

L of

aliq

uot B

for R

efer

ence

RNA

labe

ling.

Anne

aling

St

ep

2

Labe

ling

Step

5 6

6.

CyL

2 µ

ch TP Deoxynucleotide

Mix to one volume and 2 µL CyaninDeoxynu he other. S 1.

NOTE Th Mix is in en-capped tube and the Cyanine-5-dUTP Deoxynucleotid in a red-capped tube. Both are light sensitive. Minimize

Divide the Master Mix into two equal volumes. For eaarray to be run, add 2 µL Cyanine-3-dU

e-5-dUTP ee Figurecleotide Mix to t

e Cyanine-3-dUTP Deoxynucleotide a gree Mix is

light exposure.

Use Table 1 (see opposite page) to determine the amounts of reagents required for 2 or more arrays.

Purified Cyanine-5-Labeled cDNAs Purified Cyanine-3-Labeled cDNAs

Figure 1. First-strand cDNA labeling scheme

Reaction Master Mix 8 µL 5X Buffer + 4 µL DTT + 4 µL ENZ-RT™ / RNase Inhibitor

per array (2 labeling reactions)

anine-3-dUTP abeling Mix L per reaction

Cyanine-5-dUTP Labeling Mix

2 µL per reaction

Primer-Annealed Experimental RNA Primer-Annealed Reference RNA

Divide into two equal volumes

8

C.

9

SYN

7. e containing primer-annealed experimental RNA and 10 µL of Cyanine-5 reaction mix to each tube containing primer-a . C

8. In r.

9. the

ice

DEG

11. ect the reaction mix at the bottom

ube.

ION The RNase Cocktail will cause degradation of RNA.

eagent come in contact with rior to cDNA synthesis.

12.

PURIFICATI

In order to maximize hybrid nal and minimize non-specific hybimportant degradat reaction

THESIZE CYANINE-3- AND CYANINE-5-LABELED CDNAS Aliquot 10 µL of Cyanine-3 reaction mix to each tub

nnealed reference RNA. Mix by pipeting up and downentrifuge briefly.

cubate the cDNA labeling reactions at 42°C for 1 hou

Transfer the reaction tubes to a 70°C water bath orheating block. Incubate for 10 minutes to inactivate reverse transcriptase and RNase Inhibitor.

10. Cool the reaction tubes on ice for 2 minutes. Keep onuntil ready for the next step.

RADE THE RNA TEMPLATE

Briefly centrifuge to collof the tube. Then add 2 µL RNase Cocktail to each reaction.

NOTE The RNase Cocktail is in a yellow-capped t

CAUT

Be careful not to let this rexperimental or reference RNA p

Incubate at 37°C for 30 minutes.

ON OF LABELED CDNAS

ization siground on mridization backg icroarrays, it is

to purify the fluorescent cDNA probes after i g separately. on of the RNA template. Purify each labelin

D.

We recommend using Qiagen’s QIAqupurifying labeled cDNA samples. Thes

ick® spin columns for e columns efficiently

remove free nucleotides including unincorporated dye-labeled cleotides. Follow the protoco ow. All

00 rpm).

tube. e it

ame

7. centrifuge for

on and yield, see section D, page 10.

Y

The f labe uantified by UV/visible spectrophoto-metry. Determination of both inco ration and yield can be most qui in thelabeled cDNelution buffer EB to the column eluates of each labeled cDNA sample for spectrophotometric analysis of the entire sample (e.g. 80 µL in a 50 µL cuvette).

nu l described belcentrifuge steps are at ≥10,000 x g (~13,0

1. Add 250 µL of Qiagen Buffer PB to each reaction mix.

2. Place a QIAquick® spin column in a 2 ml collection Then apply the sample onto the column and centrifugin a tabletop centrifuge for 30 seconds.

3. Discard flow-through and place the column back into the same collection tube.

4. Wash the column with 750 µL of Qiagen Buffer PE and centrifuge for 30 seconds.

5. Discard flow-through, place the column back into the scollection tube and centrifuge for 2 minutes to dry the column.

6. Transfer the column into a clean 1.5 ml microcentrifuge tube.

To elute the sample, add 25 µL of Qiagen Buffer EB to the center of the column, let sit for 1 minute, then 2 minutes. Repeat this step with another 25 µL of Qiagen Buffer EB.

NOTE For determination of incorporati

For size distribution analysis, see section E, page 11.

DETERMINATION OF INCORPORATION AND YIELD OF C ANINE-LABELED NUCLEOTIDE

incorporation of cyanine-labeled nucleotides and yield oled cDNA can be q

rpo Nanodckly assessed using a rop™ spectrophotometer

Microarray mode with 1- 2 µL of column eluate for each A sample. Alternatively, add a defined volume of

10

11

CALCULATION OF INCORPORATION m ts

for cyanine-5, the

n reaction, purified and

TP

ALCULATION OF YIELD he yield of labeled cDNA can be estimated by taking bsorbances at 260 nm and calculating as follows, given that n A260 of 1 equals 33 µg/mL single-stranded DNA:

CALCULAT

The frequfollows:

OI is the umber of cyanine-labeled nucleotides per 1000 bp cDNA. In eneral, the FOIs for cyanine-labeled samples to be

ected to microarray hybridization should be > 10 but < 70.

Cyanine-3 and cyanine-5 have absorption maxima at 550 nand 650 nm respectively. Using molar extinction coefficienof 150,000 for cyanine-3 and 250,000 picomoles of cyanine-labeled nucleotide per reaction can be calculated as follows:

Example: A cyanine-5 incorporatioeluted in 80 µL (for reading in a 50 µL cuvette), givesan undiluted absorbance at 650 nm of 0.139. This corresponds to 44.5 total pmoles cyanine-5-dUincorporated.

C

Taa

ION OF FREQUENCY OF INCORPORATION (FOI) ency of incorporation (FOI) may be calculated as

n an average cDNA length of 1000 bp, the FBased ongsubj

FOI =ng cDNA

pmols cyanine-dUTP incorporated x 324.5

= µg cDNA 1,000

A260 x 33 x volume (µL)

x volume (µL) x 106 = pmols Cyanine-5 A650

250,000

x volume (µL) x 106 = pmols Cyanine-3 150,000

A550

E. SIZE DISTRIBUTION ANALYSIS BY GEL

nce of the cyanine-labeled cDNA. . Remove 2-5 µL from each of the column eluates (prior to

combining the labeled cDNAs) and analyze on 1-2% agarose gel. Include a DNA size standard. Use the smallest available gel. Prepare the cyanine-labeled cDNA and the DNA size standard with minimal loading buffer.

10,000 in running for SYBR® Gold signals from both the

e two reactions.

F. PRHY

-a

a ce

CAUTION

conditions and temperatures. We recommend using the hybridization buffer supplied or suggested by the microarray

ELECTROPHORESIS

Agarose gel electrophoresis can be used to visualize the fluoresce1

2. Scan the gel using an apparatus that can detect both the cyanine-3 and cyanine-5 lanes. The emission wavelengthsof cyanine-3 and cyanine-5 are 570 nm and 670 nm, re ectively. sp

3. Stain the gel with SYBR® Gold stain (1:buffer). Then, scancyanine-3 and cyanine-5 bands.

4. Compare the DNA size distribution for th

EPARATION OF LABELED CDNA FOR MICROARRAY BRIDIZATION

After column purification, combine the cyanine-3- and cyanine5-l beled cDNAs, freeze in dry ice and lyophilize to dryness in

ntrifugal concentrator.

The cyanine-labeled cDNAs are light sensitive. Be sure to protect from light during lyophilization.

Different microarray manufacturers utilize various hybridization

manufacturer at a volume sufficient for the dimensions of the cover slip.

If the labeled cDNAs are not to be used within 2 weeks, store the lyophilized labeled cDNAs at -80°C until ready for use. For immediate use, proceed to the following steps:

12

1.

2.

G.

MA

3.

PR

If nbe prothe

DE

13

Add the appropriate vo ybridization buffer to the lyop

Heat the solu 0 minutes.

T

) supplied in this kit contains 1 µg/µL of a 1,200 bp poly A+ mRNA.

50 µL)

e gel and apparatus

A and tube B), combine 1 µLOligo dT Primer (dT), 2 µL) Control RNA (C) and 7 µL for 5 minutes

Deoxynucleotide Mix Cyanine-5

(B), 2 µL 10X DTT and (RT) to each tube.

lume of hhilized labeled cDNAs. Vortex thoroughly.

tion at 70°C in a water bath for 5-1

Maintain the probe solution at hybridization temperature while preparing arrays.

OCEDURE VALIDATION

ecessary, the Control RNA (C) supplied with this kit shouldused to confirm that all components of the kit are functioning perly and that the procedure is performing satisfactorily in user’s hands.

ERIALS NEEDED

• Control RNA (C) (supplied)

NOTE The Control RNA (C

• Small volume cuvette (≤

• QIAquick® purification columns and reagents

• UV/visible spectrophotometer

• 1-2% agaros

TAILED PROCEDURE

1. In two separate tubes (tube 2 µg (

Nuclease-free Water (W). Heat at 70°C, then place on ice for 2 minutes.

2. To tube A, add 2 µL Cyanine-3(green-capped) and to tube B, add 2 µL Deoxynucleotide Mix (red-capped).

3. Add 4 µL 5X Reaction Buffer™2 µL ENZ-RT / RNase Inhibitor

4.

6.

5.

7.

RECOMMEND1. Us

m

2. Windisp

3. Haco

A

Incubate at 42°C for 1 hour.

at 70°C for 10 minutes, then add 2 µL

to

See page 11 for

Heat inactivate RNase Cocktail (yellow capped) and incubate at 37°C for 30 minutes. Column purify the reactions as described on pages 9-10.

Measure absorbance of the undiluted eluates at 260 nmto determine cDNA yield, then at 550 nm or 650 nm determine Cyanine-dye incorporation. calculations.

Below are typical results from the above experiment:

cDNA Yield (µg) pmols

Cyanine-3 0.868 ± 0.059 94.3 ± 4.5 Cyanine-5 0.717 ± 0.053 42.0 ± 5.7

i

e recomme from Erie Scientific fferent sizes and form a three-dimensional hybridization a r the arrayed DNA spots.

ndl ay and , mpr to lint area.

TIONS FOR HYBRIDIZATION e the hybridization buffer supplied or suggested by the croarray manufacturer.

nd the use of Lifter Slips™ place of regular cover slips. These are available in

ce ove

e microarr s with gloved h s. Use dryessed air clean dust and from array

14

1

TROUBLESHOOTING GUIDE PROBLEM POSSIBLE CAUSE SOLUTION

One or more components wasout of the reaction.

Repeat the reaction with left all components included.

RNA is contaminated. Perform all necessary precautions to prevent RNase contamination. Re-isolate the RNA if necessary.

Low quality of RNA template

Confirm purity of RNA preparation. If significant degradation is observed, re-isolate. See page 5 for RNA Analysis.

Low cDNA yield

Insufficient quantity of A

Re-quantif

reaction.

prime-able RNtemplate.

y experimental RNA and/or increase amount used in labeling

Predominant cDNA size is <300 bases in length.

is contaminated with RNases or inhibitors of reverse transcriptase.

RNA starting material Re-isolate RNA and

confirm integrity on gel.

Reaction tubes may have been exposed to light.

Protect from light duringsynthesis and purification. Use amber-colored tubes whenever possible.

Low fluorescence of cDNA product

Contamination with mmon

T, reducing agents

Keep clear of colaboratory reducing agents(βME, high levels of DTetc.) during synthesis

Insufficient ethanol riate volume of e

wash buffer and keep tightly closed

was added to wash buffer.

Add appropabsolute ethanol to thfreshly opened bottle of

Columns were not Spin columns with empty t spun dry prior to

elution. collection tubes for at leas2 minutes.

Low recovery of

n application of elution buffer onto f the

labeled cDNAfrom column

Uneve

column

Carefully apply elution buffer to the center ocolumn bed such that buffer contacts the entire bed.

5

REFERENicroa Molecular C

David Bowtell and Joseph Samb pring Harbor Laboratory Pre

CES 1. DNA M rrays: A loning Manual, Eds.

rook, Cold Sss, 2003.

16

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