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LETTERSINBIOTECHNOLOGYVol．20No．4July,2009doi:10．3969／j．issn．1009－0002．2009．04．041PCR1,2，1,21．，100853；2．302，100039［］PCRmRNA，，PCRPCR。PCR，mRNA。PCR。［］PCR；；［］Q789［］A［］1009－0002(2009)04－0598－03DevelopmentandDataAnalysisofReal－TimeFluorescentQuantitativePCRJIDong1,2,XINShao－jie1,21．MilitaryPostgraduateMedicalSchool,Beijing100853;2．The302HospitalofPLA,Beijing100039;China［Abstract］Thereal－time,fluorescence－basedreversetranscriptionpolymerasechainreactionisoneoftheenablingtech-nologiesofthegenomicageandhasbecomethemethodofchoiceforthedetectionofmRNA,playsimportantrolesinclinicalexaminationsandbasictheoryresearch．Twodifferentmethodsofpresentingquantitativegeneexpressionareabso-luteandrelativequantification．Severalfactorshavecontributedtothetransformationofthistechnologyintoamainstreamresearchtool:itavoidstheneedforpost－PCRprocessing;ithasawidedynamicrangewhichallowsstraightforwardcom-parisonbetweenRNAsthatdifferwidelyintheirabundance;anditrealisestheinherentquantitativepotentialofthePCR,makingitaquantitativeaswellasqualitativeassay．Inthisarticle,thedevelopmentanddataanalysisofreal－timefluo-rescentquantitativePCRwereoverviewed．［Keywords］real－timePCR;quantification;methodology1983KaryMullis（polymerasechainreaction，PCR），。PCR，（real－time）PCR，[1－4]。1PCR1．1、、。，、、、。mRNA，[5]。，，Northern／Southern、（HPLC）、PCR－ELISA、RNA、。、、、[6]。PCR：①DNA、（EB），；②，PCR、，DNA。，，，。，（／）[7]。PCR。PCR，。TaqDNA5＇→3＇（fluorescenceresonanceenergytransfer，FRET）[8]，PCR，，。PCR。1．2PCRPCRPCR（），PCR。DNA，，PCR。5＇（R），3＇（Q），［］2008－12－01［］（30700713）［］（1975－），，，［］，（E－mail）xsj302＠yahoo．com．cn598，5＇，，，，。：，（cyclethreshold，CT）。2：①：PCR，，15；②CTPCR。，PCR[9]（1）。1．3PCRPCR（5log）。：①，5；②；③、；④，PCR，。PCR：①PCR，PCR；②RNA；③PCR，，。，，；，／；；mRNA，[10]。1．4PCRPCR，、[11－13]。1．4．1／，，PCR。，CT，，，CT，。DNARNA。。1．4．2DNAPCR，，、。，，，，PCR。，PCR（1000）。，，““（）“”[14]。1．4．3、、PCRDNA，[15]。1．4．4（SNP），，。，，，PCR。[16－17]。2PCR2．1，PCR。，（HBVDNA），，、。，，，，。，[18－19]。，。，CT，，t（ANOVA）。CT，。CT2－CT，。CT（2－ΔΔCT）。2：①100％：PCR，；②。：①；②；③，CT；④：，。：①（foldchange）＝2－ΔΔCT；②ΔΔCT＝（CT－CT）－（CT－CT）[20－23]。2．2PCR，，。，1。，。：①；②；③。，。3－（GAPDH）、β－、β2－、18SRNA、－6－（G6PDH）[24]。，，（basketapproach），，，。Basket。。，RNA。1PCRPCRSYBRGreenTaqmanLightcycleLUXAppliedBiosystemsRocheSigma－Aldrich，IDTInvitrogenDNA：PCR599LETTERSINBIOTECHNOLOGYVol．20No．4July,2009RNA，，PCR，。，RNA1，、、，PCR，RNA。RNA，RNA，[25－26]。2．3／PCR／，，。，，，／。PrimerBank（http:∥pga．mgh．harvard．edu／primerbank）、QPDO（http:∥web．ncifcrf．gov／rtp／geVprimerdb）。3PCR25，PCR20，、，（）。PCR。[1]GuanH,YangK．RNAisolationandreal－timequantitativeRT－PCR[J]．MethodsMolBiol,2008,456:259．[2]ZimmermannK,MannhalterJW．TechnicalaspectsofquantitativecompetitivePCR[J]．Biotechniques,1996,21(2):268－279．[3]FreemanWM,WalkerSJ,VranaKE．QuantitativeRT－PCR:pitfallsandpotential[J]．Biotechniques,1999,26(1):112－125．[4]AryaM,ShergillIS,WilliamsonM,etal．Basicprinciplesofre-al－timequantitativePCR[J]．ExpertRevMolDiagn,2005,5(2):209－219．[5]BustinSA．Real－time,fluorescence－basedquantitativePCR:asnapshotofcurrentproceduresandpreferences[J]．ExpertRevMolDiagn,2005,5(4):493－498．[6]HoebeeckJ,SpelemanF,VandesompeleJ．Real－timequantitativePCRasanalternativetoSouthernblotorfluorescenceinsituhy-bridizationfordetectionofgenecopynumberchanges[J]．MethodsMolBiol,2007,353:205－226．[7]ProvenzanoM,MocellinS．Complementarytechniques:validationofgeneexpressiondatabyquantitativerealtimePCR[J]．AdvExpMedBiol,2007,593:66－73．[8]WongML,MedranoJF．Real－timePCRformRNAquantitation[J]．Biotechniques,2005,39(1):1－11．[9]AhmadAI,GhasemiJB．NewFRETprimersforquantitativere-al－timePCR[J]．AnalBioanalChem,2007,387(8):2737－2743．[10]VanGuilderHD,VranaKE,FreemanWM．Twenty－fiveyearsofquantitativePCRforgeneexpressionanalysis[J]．Biotechniques,2008,44(5):1619－1626．[11]EspyMJ,UhlJR,SloanLM,etal．Real－timePCRinclinicalmicrobiology:applicationsforroutinelaboratorytesting[J]．ClinMi-crobiolRev,2006,19(1):165－256．[12]WatzingerF,EbnerK,LionT．Detectionandmonitoringofvirusinfectionsbyreal－timePCR[J]．MolAspectsMed,2006,27(2－3):254－298．[13]KaltenboeckB,WangC．Advancesinreal－timePCR:applicationtoclinicallaboratorydiagnostics[J]．AdvClinChem,2005,40:219－259．[14]ShihSC,SmithLE．Quantitativemulti－genetranscriptionalpro-filingusingreal－timePCRwithamastertemplate[J]．ExpMolPathol,2005,79(1):14－22．[15]Mazet－WagnerAA,BacletMC,Loustaud－RattiV,etal．Real－timePCRquantitationofhepatitisBvirustotalDNAandcova-lentlyclosedcircularDNAinperipheralbloodmononuclearcellsfromhepatitisBvirus－infectedpatients[J]．JVirolMethods,2006,138(1－2):70－79．[16]ShihYH,YehSH,ChenPJ,etal．HepatitisBvirusquantifi-cationanddetectionofYMDDmutantsinasinglereactionbyre-al－timePCRandannealingcurveanalysis[J]．AntivirTher,2008,13(4):469－480．[17]WelzelTM,MileyWJ,ParksTL,etal．Real－timePCRassayfordetectionandquantificationofhepatitisBvirusgenotypesAtoG[J]．JClinMicrobiol,2006,44(9):3325－3333．[18]ValasekMA,RepaJJ．Thepowerofreal－timePCR[J]．AdvPhysiolEduc,2005,29(3):151－159．[19]BustinSA,BenesV,NolanT,etal．Quantitativereal－timeRT－PCR－aperspective[J]．JMolEndocrinol,2005,34(3):597－601．[20]SchmittgenTD．Real－timequantitativePCR[J]．Methods,2001,25(4):383－385．[21]SchmittgenTD,ZakrajsekBA,MillsAG,etal．Quantit