ProtocolforReal-TimeRT-PCRThisprotocoldescribesthedetailedexperimentalprocedureforreal-timeRT-PCRusingSYBRGreenIasmentionedinXiaoweiWangandBrianSeed(2003)APCRprimerbankforquantitativegeneexpressionanalysis.NucleicAcidsResearch31(24):e154;pp.1-8.PleaserefertothispaperandthePrimerBankHelppageformorebackgroundinformation.ThisprotocolisalsoavailableinHYPERLINK"http://pga.mgh.harvard.edu/primerbank/PCR_protocol.doc"\t"_blank"MicrosoftWordformathere.HereisaHYPERLINK"http://pga.mgh.harvard.edu/primerbank/PCR_protocol_short.doc"\t"_blank"one-pagesuccinctprotocol.TheprocedurebeginswithreversetranscriptionoftotalRNA.ThecDNAisthenusedastemplateforreal-timePCRwithgenespecificprimers.Youmayneedtomodifythisprotocolifyouusedifferentreagentsorinstrumentsforreal-timePCR.TimerequiredcDNAsynthesis:2hours.real-timePCR:2hours.Dissociationcurveanalysis:0.5hour.ReagentsandEquipmentsOligonucleotidePrimers.GenespecificprimersareretrievedfromPrimerBank(HYPERLINK"http://pga.mgh.harvard.edu/primerbank/"\t"_blank"http://pga.mgh.harvard.edu/primerbank/).TheseprimersareorderedfromtheMGHDNACorefacility(HYPERLINK"https://dnacore.mgh.harvard.edu/synthesis/index.shtml"\t"_blank"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).DetailedprocedureReverseTranscriptionReverseTranscriptioniscarriedoutwiththeSuperScriptFirst-StrandSynthesisSystemforRT-PCR.ThefollowingprocedureisbasedonInvitrogen’sprotocol.1.PreparethefollowingRNA/primermixtu