ProtocolforcompetitiveRT-PCR 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). Figure1:MeasurementoftwoRNAsamples.Thelowerbandsarethestandardbands,theupperbandsarethetranscriptbands.Lane1-4:sample3;lane5-8:sample4;lane1+4:250pgstandard;lane2+5:100pgstandard;lane3+6:50pgstandard;lane4+8:25pgstandard.TherectanglesrepresentobjectssetwiththeImageQuantsoftware. Figure2:Graphicalrepresentationoftheratiosmeasuredabove Inthisexample,sample3contains55pgofthemRNAinquestion(inourcasetheNF1mRNA)andsample486pg.Assaidbefore,theseabsolutevaluesarenotreliable.Inthiscase,wesetonevalue(e.g.thecontrolorthevalueunderonecondition)to100%(inthiscasesample4)andsimplyrelatetheothervaluetothis.Sotheconclusiondrawnfromthismeasurementisthatsample3(RNAfromcellsofaNF1patient)containsonly64%ofthemRNAamountassample4(RNAfromcellsofahealthycontrol). Literature CeliFS,ZenilmanME,ShuldinerAR.1993:ArapidandversatilemethodtosynthesizeinternalstandardsforcompetitivePCR.NucleicAcidsResearch21;p.1047 SchneebergerC,SpeiserP,KuryF,ZeillingerR.1995:Quantitativedetectionofreversetranscriptase-PCRproductsbymeansofanovelandsensitiveDNAstain.PCRMethodsandApplications4;p.234-238 QuantitativeRT-PCR.MethodsandApplicationsBook3;ClontechLaboratories,Inc. TechnicalTipsOnline:Nr.01214:Eferletal.:EvaluationofdifferentRNAstandardsforreversetranscriptionPCR
ThebandintensitiesarethenanalyzedbyImageQuantsoftware(MolecularDynamics)asfollows:placerectangularobjectsovereachbandwithalittlebackground(whichissettolocal)andstart„VolumeReport".Theratiosoftranscripttostandardbandintensity(volumetranscript/volumestandard)arethenfixedinadoublelogarithmicgraphicalrepresentation(HarvardGraphics,ExcelorMicrocalOrigin)inwhichtheamountofstandardmRNAequaltotheamountoftranscriptRNAcanbereadattheintersectionofthelinewiththey-value1.Ideally,the4measuredratiosshouldyieldastraightline.However,becauseofvariancesduetopipetting,dilutionetc,theratiosoftenamounttoaslightzig-zag.Inthiscase,aregressionlineiscalculatedordrawn.
