ProtocolforReal-TimeRT-PCR

Thisprotocoldescribesthedetailedexperimentalprocedureforreal-timeRT-PCRusingSYBRGreenIasmentionedinXiaoweiWangandBrianSeed(2003)APCRprimerbankforquantitativegeneexpressionanalysis.NucleicAcidsResearch31(24):e154;pp.1-8.PleaserefertothispaperandthePrimerBankHelppageformorebackgroundinformation.ThisprotocolisalsoavailableinMicrosoftWordformathere.Hereisaone-pagesuccinctprotocol.TheprocedurebeginswithreversetranscriptionoftotalRNA.TheCDNAisthenusedastemplateforreal-timePCRwithgenespecificprimers.Youmayneedtomodifythisprotocolifyouusedifferentreagentsorinstrumentsforreal-timePCR.

	cDNAsynthesis:2hours.
	real-timePCR:2hours.
	Dissociationcurveanalysis:0.5hour.

	OligonucleotidePrimers.GenespecificprimersareretrievedfromPrimerBank(http://pga.mgh.harvard.edu/primerbank/).TheseprimersareorderedfromtheMGHDNACorefacility(https://dnacore.mgh.harvard.edu/synthesis/index.shtml).AlltheprimersaredesaltedandbothUVabsorbanceandcapillaryelectrophoresisareusedtoassessthequalityofprimersynthesis.
	MousetotalliverRNA(Stratagene).
	MousetotalRNAmasterpanel(BDBiosciences/Clontech).
	SYBRGreenPCRmastermix,200reactions(AppliedBiosystems).
	Opticaltubeandcapstrips(AppliedBiosystems).
	SuperScriptFirst-StrandSynthesisSystemforRT-PCR(Invitrogen).
	25bpDNAladder(Invitrogen).
	ABIPrism7000SequenceDetectionSystem(AppliedBiosystems).
	ABIPrism7000SDSsoftware(AppliedBiosystems).
	3%ReadyAgarosePrecastGel(Bio-Rad).
	Agarosegelelectrophoresisapparatus(Bio-Rad).

ReverseTranscription

ReverseTranscriptioniscarriedoutwiththeSuperScriptFirst-StrandSynthesisSystemforRT-PCR.ThefollowingprocedureisbasedonInvitrogen’sprotocol.

1.PreparethefollowingRNA/primermixtureineachtube:

2.Incubatethesamplesat65°Cfor5minandthenoniceforatleast1min.3.Preparereactionmastermixture.Foreachreaction:

4.AddthereactionmixturetotheRNA/primermixture,mixbriefly,andthenplaceatroomtemperaturefor2min.5.Add1ml(50units)ofSuperScriptIIRTtoeachtube,mixandincubateat25°Cfor10min.6.Incubatethetubesat42°Cfor50min,heatinactivateat70°Cfor15min,andthenchillonice.7.Add1mlRNaseHandincubateat37°Cfor20min.8.Storethe1ststrandcDNAat-20°Cuntiluseforreal-timePCR.

Real-timePCR

1.Normalizetheprimerconcentrationsandmixgene-specificforwardandreverseprimerpair.Eachprimer(forwardorreverse)concentrationinthemixtureis5pmol/ml.2.SetuptheexperimentandthefollowingPCRprogramonABIPrismSDS7000.DonotclickonthedissociationprotocolifyouwanttocheckthePCRresultbyagarosegel.SaveacopyofthesetupfileanddeleteallPCRcycles(usedforlaterdissociationcurveanalysis).Pleasenotetheextensionstepsareslightlydifferentfromdescribedinourpaper.

1)50°C2min,1cycle2)95°C10min,1cycle3)95°C15s->60°C30s->72°C30s,40cycles4)72°C10min,1cycle

3.Areal-timePCRreactionmixturecanbeeither50mlor25ml.Preparethefollowingmixtureineachopticaltube.

4.AfterPCRisfinished,removethetubesfromthemachine.ThePCRspecificityisexaminedby3%agarosegelusing5mlfromeachreaction.5.PutthetubesbackinSDS7000andperformdissociationcurveanalysiswiththesavedcopyofthesetupfile.6.Analyzethereal-timePCRresultwiththeSDS7000software.Checktoseeifthereisanybimodaldissociationcurveorabnormalamplificationplot.

HereIlistedafewmajorcausesforreal-timePCRfailures.PleasereadthePrimerBankHelppageformoredetails.LittleornoPCRproduct.PoorqualityofPCRtemplates,primers,orreagentsmayleadtoPCRfailures.First,pleaseincludeappropriatePCRcontrolstoeliminatethesepossibilities.Somegenesareexpressedtransientlyoronlyincertaintissues.Inourexperience,thisisthemostlikelycausefornegativePCRresults.Pleasereadliteratureforthegeneexpressionpatterns.Onecaveatisthatmicroarraysarenotalwaysreliableatmeasuringgeneexpressions.Afterswitchingtotheappropriatetemplates,weobtainedpositivePCRresultsincontrasttotheotherwisenegativePCRs(seeourpaperformoredetails).PoorPCRamplificationefficiency.Theaccuracyofreal-timePCRishighlydependentonPCRefficiency.Areasonableefficiencyshouldbeatleast80%.PoorprimerqualityistheleADIngcauseforpoorPCRefficiency.Inthiscase,thePCRamplificationcurveusuallyreachesplateauearlyandthefinalfluorescenceintensityissignificantlylowerthanthatofmostotherPCRs.Thisproblemmaybesolvedwithre-synthesizedprimers.Primerdimer.Primerdimermaybeoccasionallyobservedifthegeneexpressionlevelisverylow.Ifthisisthecase,increasingthetemplateamountmayhelpeliminatetheprimerdimerformation.Multiplebandsongelormultiplepeaksinthemeltingcurve.AgarosegelelectrophoresisormeltingcurveanalysismaynotalwaysreliablymeasurePCRspecificity.Fromourexperience,bimodalmeltingcurvesaresometimesobservedforlongamplicons(>200bp)evenwhenthePCRsarespecific.Theobservedheterogeneityinmeltingtemperatureisduetointernalsequenceinhomogeneity(e.g.independentlymeltingblocksofhighandlowGCcontent)ratherthannon-specificamplicon.Ontheotherhand,forshortamplicons(<150bp)veryweak(andfussy)bandsmigratingaheadofthemajorspecificbandsaresometimesobservedonagarosegel.Theseweakbandsaresuper-structuredorsingle-strandedversionofthespecificampliconsinequilibriumstateandthereforeshouldbeconsideredspecific.Althoughgelelectrophoresisormeltingcurveanalysisalonemaynotbe100%reliable,thecombinationofbothcanalwaysrevealPCRspecificityinourexperience.Non-specificamplicons.Non-specificamplicons,identifiedbybothgelelectrophoresisandmeltingcurveanalysis,givemisleadingreal-timePCRresult.Toavoidthisproblem,pleasemakesuretoperformhot-startPCRanduseatleast60°Cannealingtemperature.Wenoticednotallhot-startTaqpolymerasesareequallyefficientatsuppressingpolymeraseactivityduringsamplesetup.TheSYBRGreenPCRmastermixdescribedherealwaysgivesussatisfactoryresults.Ifthenon-specificampliconispersistent,youhavetochooseadifferentprimerpairforthegeneofinterest.YouarealsoencouragedtoreportbadprimerstoXiaoweiWang(xwang@molbio.mgh.harvard.edu).

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