(thisprotocolwasadaptedbyJoeDeRisifromonedevelopedatRosettaInpharmatics)

ResUSPendthecontentsofonepackofthemonofunctionalNHS-esterCy3orCy5dye(mono-functionalCy3orCy5reactivepack,AmershamcatalogPA23001andPA25001)in72ulDMSO.Aliquot16x4.5ulandeitheruseimmediatelyordrythealiquotsinaspeedvac.Storealiquotsat4Cinadessicator.UseonealiquotofthedyesforeachRTlabellingreactionandmicroarrayhybridization.

I.RTReaction

OligodT/RandomPrimeRNA:

CDNAsynthesis

II.Hydrolysis

Add:10ul1NNaOH

10ul.5MEDTA

Incubate:15min.at65°C.

Neutralize:25ul1MTrispH7.4or1MHEPESpH7.5

III.CleanupTocontinuewiththeamino-allyldyecouplingprocedureallTrismustberemovedfromthereactiontopreventthemonofunctionalNHS-esterCyedyesfromcouplingtofreeaminegroupsinsolution.

FilloneMicrocon30concentratorwith450ulwater.

Addneutralizedreaction.

Spinat12Kfor8minutes.

Dumpflo-thru.

Repeatprocess2X,refillingoriginalfilter.

Elute.

Dryeluateinspeedvac.

IV.Coupling

ResuspendcDNApelletin9ul0.1MNABIcarbonateBufferpH9.0.AddthistothedriedaliquotofCy3orCy5dye.MixdyeandcDNA.

Letincubate1houratRTindark.

V.QuenchingandCleanupBeforecombiningCy3andCy5samplesforhybridizations,thereactionsmustbequenchedtopreventcross-coupling.

Add4.5ul4Mhydroxylamine.

Letreactionincubate15min.atRTindark.

Toremoveunicorporated/quenchedcyedyesproceedwithQia-quickPCRpurificationkit.

CombineCy3andCy5reactions.

Add70ulwater.Add500ulBufferPB.

ApplytoQia-quickcolumnandspinat13,000rpminfor30-60sec.

Aspirateoffflo-thru.

Add750ulBufferPEandspin30-60sec.

Aspirateoffflo-thruandrepeat.Aspirateflo-thruandspinfor1min.athighspeedtodrycolumn.

Transfertofreshepp.tube

Add30ulBufferEBtocenteroffilterandletsit1min.atRT.

Spinat13,000rpmfor1min.

Repeatelutionstepagain.

VI.Hyb.Prep.

DrydownQia-quickeluateinspeedvac.

Bringvolumeto18ulwithwaterandHEPESpH7.5sothatthefinalconcentration(afterSSCandotheradditions)is25mM.

Add:3.6ul20XSSC

1.8ulpolyA(10mg/ml)and/orothercompetitornucleicacid

Optional:filterinMillipore0.45micronspincolumn.Add:0.54ul10%SDS.

Incubatereactionat100°Cfor2min.

Applytopreparedmicroarray.

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