Simulation of between Repeat Variability in Real Time PCR Reactions 英文参考文献.docVIP

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Simulation of between Repeat Variability in Real Time PCR Reactions 英文参考文献.doc

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Simulation of between Repeat Variability in Real Time PCR Reactions 英文参考文献

SimulationofbetweenRepeatVariabilityinRealTime PCRReactions AntoonLievens1,2*,StefanVanAelst2,MarcVandenBulcke1,3,ElsGoetghebeur2 1PlatformforMolecularBiologyandBiotechnology,ScientificInstituteofPublicHealth,Brussels,Belgium,2DepartmentofAppliedMathematicsandComputerScience, GhentUniversity,Gent,Belgium,3MolecularBiologyandGenomicsUnit,EuropeanCommission-JointResearchCentre,InstituteforHealthandConsumerProtection, Ispra,Italy Abstract WhilemanydecisionsrelyonrealtimequantitativePCR(qPCR)analysisfewattemptshavehithertobeenmadetoquantify boundsofprecisionaccountingforthevarioussourcesofvariationinvolvedinthemeasurementprocess.Besidesinfluences ofmoreobviousfactorssuchascameranoiseandpipettingvariation,changingefficiencieswithinandbetweenreactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurementerrorandothersourcesofvariationastheycontributetofluorescenceobservationsduringtheamplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generatingrealisticvariationpatterns.Tothisend,westartfromarelativelysimplestatisticalmodelfortheevolutionof efficiencyinasinglePCRreactionandintroduceadditionalerrorcomponents,oneatatime,toarriveatstochasticdata generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variationinCq valueswasadequatelycapturedbythestatisticalmodelintermsofforeseencomponents.Torecreatethe dispersion of the repeats’ plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additionalsourcesofreagentconsumption(sidereactions)enterintothemodel.Onceanadequatedatageneratingmodel isavailable,simulationscanservetoevaluatevariousaspectsofPCRundertheassumptionsofthemodelandbeyond. Citation:LievensA,VanAelstS,VandenBulckeM,GoetghebeurE(2012)SimulationofbetweenRepeatVariabilityinRealTimePCRReactions.PLoSONE7(11): e47112.doi:10