Analysis-of-Relative-Gene-Expression-Data-Using-Re

METHODS25,402–408(2001)doi:10.1006/meth.2001.1262,availableonlineat*andThomasD.Schmittgen†,1*AppliedBiosystems,FosterCity,California94404;and†DepartmentofPharmaceuticalSciences,CollegeofPharmacy,WashingtonStateUniversity,Pullman,Washington99164-6534ofthetargetgenerelativetosomereferencegroupThetwomostcommonlyusedmethodstoanalyzedatafromsuchasanuntreatedcontrolorasampleattimezeroreal-time,quantitativePCRexperimentsareabsolutequantifica-inatime-coursestudy.tionandrelativequantification.Absolutequantificationdeter-Absolutequantificationshouldbeperformedinsitu-minestheinputcopynumber,usuallybyrelatingthePCRsignaltoastandardcurve.RelativequantificationrelatesthePCRsignalationswhereitisnecessarytodeterminetheabsoluteofthetargettranscriptinatreatmentgrouptothatofanothertranscriptcopynumber.Absolutequantificationhassamplesuchasanuntreatedcontrol.The22DDCTmethodisabeencombinedwithreal-timePCRandnumerousre-convenientwaytoanalyzetherelativechangesingeneexpressionportshaveappearedintheliterature(6–9)includingfromreal-timequantitativePCRexperiments.Thepurposeofthistwoarticlesinthisissue(10,11).Insomesituations,reportistopresentthederivation,assumptions,andapplicationsitmaybeunnecessarytodeterminetheabsolutetran-ofthe22DDCTmethod.Inaddition,wepresentthederivationandscriptcopynumberandreportingtherelativechangeapplicationsoftwovariationsofthe22DDCTmethodthatmaybeusefulintheanalysisofreal-time,quantitativePCRdata.q2001ingeneexpressionwillsuffice.Forexample,statingElsevierScience(USA)thatagiventreatmentincreasedtheexpressionofKeyWords:reversetranscriptionpolymerasechainreaction;genexby2.5-foldmaybemorerelevantthanstatingquantitativepolymerasechainreaction;relativequantification;thatthetreatmentincreasedtheexpressionofgenexreal-timepolymerasechainreaction;TaqMan.from1000copiesto2500copiespercell.Quantifyingtherelativechangesingeneexpressionusingreal-timePCRrequirescertainequations,as-sumptions,andthetestingoftheseassumptionstoReservetranscriptioncombinedwiththepolymer-properlyanalyzethedata.The22DDCTmethodmaybeasechainreaction(RT-PCR)hasproventobeapower-usedtocalculaterelativechangesingeneexpressionfulmethodtoquantifygeneexpression(1–3).Real-determinedfromreal-timequantitativePCRexperi-timePCRtechnologyhasbeenadaptedtoperformments.Derivationofthe22DDCTequation,includingquantitativeRT-PCR(4,5).Twodifferentmethodsofassumptions,experimentaldesign,andvalidationanalyzingdatafromreal-time,quantitativePCRex-tests,havebeendescribedinAppliedBiosystemsUserperimentsexist:absolutequantificationandrelativeBulletinNo.2(P/N4303859).Analysesofgeneexpres-quantification.Absolutequantificationdeterminesthesiondatausingthe22DDCTmethodhaveappearedininputcopynumberofthetranscriptofinterest,usuallytheliterature(5,6).ThepurposeofthisreportistobyrelatingthePCRsignaltoastandardcurve.Rela-presentthederivationofthe22DDCTmethod,assump-tivequantificationdescribesthechangeinexpressiontionsinvolvedinusingthemethod,andapplicationsofthismethodforthegeneralliterature.Inaddition,wepresentthederivationandapplicationoftwovaria-tionsofthe22DDCTmethodthatmaybeusefulinthe1Towhomrequestsforreprintsshouldbeaddressed.Fax:[phone].E-mail:[e-mail]-timequantitativePCRdata.4021046-2023/01$35.00q2001ElsevierScience(USA)Allrightsreserved.ANALYSISOFREAL-TIMEPCRDATA403or1.THE22DDCTMETHODXN3(11E)DCT5K,[6]1.1.Derivationofthe22DDCTMethodwhereXNisequaltothenormalizedamountoftargetTheequationthatdescribestheexponentialamplifi-(X0/R0)andDCTisequaltothedifferenceinthresholdcationofPCRiscyclesfortargetandreference(CT,X2CT,R).RearranginggivestheexpressionXn5X03(11EX)n,[1]XN5K3(11E)2DCT.[7]whereXnisthenumberoftargetmoleculesatcycleThefinalstepistodividetheXNforanysampleqbynofthereaction,X0istheinitialnumberoftargettheXNforthecalibrator(cb):molecules.EXistheefficiencyoftargetamplification,andnisthenumberofcycles.Thethresholdcycle(CT)indicatesthefractionalcyclenumberatwhichtheXN,qXN,cb5K3(11E)2DCT,qK3(11E)2DCT,cb5(11E)2DDCT.[8]amountofamplifiedtargetreachesafixedthreshold.Thus,Here2DDCT52(DCT,q2DCT,cb).Forampliconsdesignedtobelessthan150bpandforXT5X03(11EX)CT,X5KX[2]whichtheprimerandMg2+concentrationshavebeenproperlyoptimized,theefficiencyisclosetoone.There-whereXTisthethresholdnumberoftargetmolecules,fore,theamountoftarget,normalizedtoanendogenousCT,Xisthethresholdcyclefortargetamplification,andreferenceandrelativetoacalibrator,isgivenbyKXisaconstant.Asimilarequationfortheendogenousreference(internalcontrolgene)reactionisamountoftarget522DDCT.[9]RT5R03(11ER)CT,R5KR,[3]1.2.AssumptionsandApplicationsofthe22DDCTMethodFortheDDCTcalculationtobevalid,theamplificationwhereRTisthethresholdnumberofreferencemole-efficienciesofthetargetandreferencemustbeapproxi-cules,R0istheinitialnumberofreferencemolecules,matelyequal.AsensitivemethodforassessingiftwoERistheefficiencyofreferenceamplification,CT,Risampliconshavethesameefficiencyistolookathowt