Protocol for competitive RTPCR

ForquantifyingmRNA,weuseacompetitiveRT-PCRprotocolwithinternalstandardRNAs.TheseareaddedinadefinedquantitytotheRNAsamplepriortotheRTreaction.TheresultingstandardCDNAiscoamplifiedwiththesameprimersastheendogenoustargetsequence.ItsPCRproductisapproximately50nucleotidessmaller.Thismethodallowsmeasurementofsmalldifferences(aslowasfactor2)inmRNAamountbetweenRNAsamples.RNAstandardshavethebigadvantagethatalsothevariationoftheRTeffiencyisirrelevant,aswellasthevariationofthePCReffiency.

MakinginternalstandardRNAs

ThesequencetobeamplifiedshouldbestspananintronsothatgDNAcontaminationdoesnotplayacrucialrole.Otherwise,theRNAhastobetreatedwithDNaseI(RNase-free)andthesuccessofthistreatmenthastobecontrolledbyaPCRwithoutpriorRT.Itis,however,alwaysrecommendabletotreattheRNAsampleswithDNasesothatnogenomicDNAcompetesforthePCRcomponents.

Tomakeastandard,firstaPCRwithaconventionaldownstreamprimerandamodifiedupstreamprimer(40nucleotidesinlength)isperformedaccordingtoCelietal.(1993).cDNAisusedastemplateforthePCRs.ThePCRproductsareisolatedfroman1.5%agarosegelandclonedintoapGEM3ZvectorthatcontainesaT7RNApromotersequence.TheinvitrotranscriptionoftheclonedfragmentsisperformedusingT7RNApolymerase(e.g.GibcoBRL).TheinternalstandardRNAisthentreatedwithRNase-freeDNaseI(e.g.GibcoBRL)toremovetheplasmidDNA(successalsocheckedbyPCRwithoutpriorRT)andfinallyquantitatedbymeasurementoftheopticaldensityat260nmandstoredat-70°C.

ThemainproblemwithRNAstandardsistheirinstABIlity.Wefoundthatespeciallythawingandrefreezingdamagesthem.Thereforeitisbesttostorethestandardsinsmallaliquotsindifferentdilutionsanddiscardthemifthawedtoooften.Also,thisproblemmeansthatnoabsoluteamountscanbemeasured,becausethereisnowayofknowinghowmuchstandardisalreadydegradedinthealiquotused.However,thismethodisveryreliableandaccurateforcomparisonofdifferentsamplesifthesamestandardaliquotsareusedformeasuringtheirmRNAamount.

QuantitationofmRNA

ForthequantitationofthemRNAofoneRNApreparation4RTreactionsarepreparedwith1μgtotalRNAeachanddifferentamountsofstandardRNA(ifmorethanonemRNAistobequantitated,thedifferentstandardscanbemixedtogether).WeusetheSuperScriptPreamplificationSystemfromGibcoBRLforourRTs.ForthefirstmeasurementsofamRNAitisbesttoaddstandardamountswhichdifferbyfactor10(i.e.:100fg;1pg;10pg;100pg;1ng)todeterminetherangeinwhichthetranscriptamountistobefound.Ifthatisknown,factor2-2.5betweenthestandardamountsgivesmoreaccurateresults(i.e.:25pg;50pg;100pg;250pg).

InthefollowingPCR3-5μlofcDNA,1.5unitsTaqDNApolymerase(Pharmacia),200μMofeachdNTP,250nMofeachprimerand1/10volumeofa10xPCRstandardbuffer(15mMMgCl2;100mMTris/HCl,pH8.3;500mMKCl)areaddedtoatotalvolumeof50μl.ThePCRisruninthethermalcyclerGeneAmp9600(PerkinElmer).ThePCRproductsarethenseparatedona1.5%agarosegel,stainedwithethidiumbromide,SYBR-GreenorSYBR-Gold(Molecularprobes,highersensitivity,butalsomorelightsensitive)andscannedbyaCCDcamera.TheamountofcDNAusedforaPCR,thenumberofcyclesandthenucleicacidstainuseddependonhowabundantthetranscriptisthatisbeingmeasured.Ifheteroduplicesappear,onepossibiltyistorunlessPCRcycles.Iftheystillcannotbeavoided,Eferletal.addressedthisproblemextensively(TechnicalTipsOnline).