企业QQ
官方微信
RT-PCRorreversetranscriptionPCRreferstoPCRthatusesproductofanRTreactionastemplate.Ineffect,thePCRamplifiescDNAfragments.Inone-tubeRT-PCR,RNAandPCRprimersareaddedtoareactionmixthatisthermocycledforRTfirstfollowedbyforPCR.One-tubeRT-PCRreactionmixesaresuppliedbymanymanufacturers.Drawbackstotheiruseincludelackofflexibility.Otherwise,RT-PCRisatwostepprocess.StepI-RTSeeReversetranscriptionformoreonRTSpecificityofRT-PCRcanbeimprovedbyusinggenespecificprimersforRT.Itisgoodtoincludeanegativecontrolreactionintheexperiment.Thisissetuplikethetestreaction,exceptthatRTenzymeisnotaddedtoit.AbsenceofPCRproductswhenusingthecontrolreactionastemplateindicatesthatthePCRproductsdidnotarisefromgenomicDNAcontaminationoftheRNAsample.IfthePCRprimersareacrossintrons,thenthiscontrolisnotneededasPCRproductsarisingfromanygenomicDNAcontaminationwillbeoflargersizeormayevennotbeobtained(iftheintronsarelarge).StepII-PCRAfraction,usually5-20%,oftheRTproductsisdirectlyusedinPCRusinggene-specificprimers.NotesGenomicDNAcontaminationmaybeirrelavantifitgivesrisetoPCRproductsofsizesdifferentthanthosearisingfromcDNA.Otherwise,e.g.,whenprimersaredesignedfromsamegenomicexon,oneneedstotreattheRNApreparationwithDNAse(RNAse-free).TheprocessofenrichmentofmRNA(poly-A+)fromtotalRNAusingoligo-dT-affinityalsoreducesDNAcontamination.OnecanalsoaddtotheRNAsampleacombinationofrestrictionenzymestospecificallycleavegenomicDNA.Ashkenasetal.(Biotechniques39:12005)suggestmakingacocktailofAluI,NlaIII,Sau96IandStyD4I-allfrequentcutters-anddaddingittotheRTreactionat1Uea.enzyme/20ulRTreaction.BeforeRT,thereactionmixtureisincubatedat37degfor30min.Then,afterRT,thereactionistreatedat94degfor10mintokilltherestrictionenzymes.