ProtocolforReverseTranscriptionandAmino-allylCouplingofRNA Thefollowingisaslightmodification*ofaprotocoldevelopedbyJoeDeRisi(UCSF)andRosettaInpharmatics(Kirkland,WA).Originaldocumentcanbeobtainedatwww.microarrays.org. A.RTReaction 1.Toannealprimer,mix1-2mgmRNAwith5ugofanchoredoligo-dT[(dT)20-VN](Operon,HPLCpurified)inatotalvolumeof18mL.OnereactionforsamplemRNAandoneforreferencemRNA. oligodT 5mgof2.5mg/mL 2mL mRNA/water 1-2mg 16mL 2.Heatto70Cfor10minutes.Coolonicefor5minutes. 3.Add11.6mLofnucleotidemixtoeachofCy3andCy5reactions. NucleotideMixforonereaction 5XRTbuffer 50XdNTPstocksolution DTT SuperscriptIIRT(Gibco) RNasin(Gibco,optional) 0.1M 200U/mL 40U/mL 6.0mL 0.6 3.0 1.5 0.5 50XdNTPstocksolutionusinga4:1ratioaminoallyl-dUTPtodTTP***: 10mLeach100mMdATP,dGTP,dCTP(Pharmacia) 8mL100mMaminoallyl-dUTP**(Sigma,#A0410) 2mL100mMdTTP **Dissolve10mgaminoallyl-dUTPin170mLwater.Addapprox.6.8mL1NNaOH.FinalpHisroughly7.0usingpHpaper. ***Alteringtheratioofaminoallyl-dUTPtodTTPwillaffecttheincorporationofCydye. 1XdNTPfinalconcentrationduringlabeling 500mMeachdATP,dCTP,dGTP 400mMaminoallyl-dUTP 100mMdTTP 4.Incubatereactionfor1hourat42C.Addadditional1mLreversetranscriptaseandcontinueincubationat42Cforanadditional1hour. B.Hydrolysis 1.DegradeRNAbyadditionof15mLof0.1NNaOH.Incubateat70Cfor10minutes 2.Neutralizebyadditionof15mL0.1NHCl. Tocontinuewiththeamino-allyldyecouplingprocedure,allTrismustberemovedfromthereactiontopreventthemonofunctionalNHS-esterCy-dyesfromcouplingtofreeaminegroupsinsolution. 3.Add450mLwatertoeachreaction. C.Cleanup Add500mLneutralized,dilutedreactionmixtoaMicrocon-30filter(Amicon). Spinat12gfor7minutes. Discardflowthrough. Repeatprocesstwomoretimes,refillingoriginalfilterwith450mLwater.Concentrateto10mL.Samplescanbestoredat-20Cindefinitely. D.Coupling Add0.5mL1Msodiumbicarbonate,pH9.0to50mMfinal.Check1MstocksolutionperiodicallyforfluctuationsinpH. MonofunctionalNHS-esterCy3(PA23001)andCy5dye(PA25001,Amersham)issuppliedasadrypellet.Eachtubeissufficienttolabel10reactionsundernormalconditions.Dissolvedrypelletin20mLDMSO.Aliquot2mLinto10singleusetubesthatarethendriedinvacuoandstoredesiccatedat4C.NHS-esterconjugatedCydyeisrapidlyhydrolyzedinwater,therefore,donotstoreinDMSOorwater.Decreasingthenumberofaliquots/dyetubemayincreaseyoursignal. Ifyouhavealreadymadealiquotsofdye,simplytransferyourCDNAinbicarbonatebuffer(10.5mL)tothealiquotofdye.Alternatively,dissolveCydyein10ulDMSOandadd1mLofdyeto10.5mLofthecDNAreaction.10%DMSOinthecouplingreactionwillnotaffectthechemicalreaction.Aliquotunuseddyeanddryimmediately. Incubate1houratRTinthedark.Mixevery15minutes. E.QuenchingandCleanup BeforecombiningCy3andCy5samplesforhybridization,unreactiveNHS-esterCydyemustbequenchedtopreventcrosscoupling. Add4.5mL4Mhydroxylamine(Sigma). Letreactionincubate15minutesinthedark. Toremoveunincorporated/quenchedCydyes,proceedwithQia-QuickPCRpurificationkit(Qiagen).Methoddescribedbelowisasspecifiedbymanufacturer. CombineCy3andCy5reactions. Add70mLwater. Add500mLBufferPB. ApplytoQia-quickcolumnandspinat13Kfor30-60seconds.(optional:reapplyflow-thoughforoptimalbinding). Decantflow-through. Add750mLBufferPEandspin30-60seconds. Decantflow-through. Spinathighspeedtodrycolumn. TransferspinunittofreshEppendorftube. Add30mLBufferEBtocenteroffilterandallowtosit3minutesatRT. Spinat13Krpmfor1minute. Repeatelutionstepagainwithanother30mLofBufferEB. Pooleluates. Add20mL(20mg)CotDNA(Gibco). Add420TEandapplytofreshMicrocon-30filter. Spin12,000gtoavolumeof29mLorless. For38mLarrayhybridization: 29mLcDNAprobeinTE 1mLpolyA(10mg;SigmaP9403) 1mLtRNA(10mg;Gibco#15401-029) 7mL20XSSC 1.2mLSDS10% Heatto100Cfor2minutes.Letstand15minutesRT. Apply38mLto40Karray. *SlightmodificationstooriginalprotocolbyMitchGarberandAnatolyUrisman.
