Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves 英文参考文献.docVIP

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Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves 英文参考文献.doc

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Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves 英文参考文献

Real-TimeRelativeqPCRwithoutReferencetoControl SamplesandEstimationofRun-SpecificPCRParameters fromRun-InternalMini-StandardCurves JensMagnusBernthJensen1*,MikkelSteenPetersen1,MarcStegger2¤,LarsJ.?stergaard2,BjarneK. M?ller1 1Department of Clinical Immunology, University Hospital of Aarhus, Skejby, Denmark, 2Department of Infectious Diseases, University Hospital of Aarhus, Skejby, Denmark Abstract Background:Real-TimequantitativePCRisanimportanttoolinresearchandclinicalsettings.Here,wedescribetwonew approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and providebothrun-specificandtarget-specificestimatesofPCRparameters.ThedrqPCRenablesdirectestimationoftarget ratioswithoutreferencetoconventionalcontrolsamples. Methodology/PrincipalFindings:Inthisstudy,wecomparedRIMS-baseddrqPCRwithclassicalquantificationsbasedon externalstandardcurvesandthe‘‘comparativeCtmethod’’.Specifically,weusedarawreal-timePCRdatasetasthebasisfor morethantwo-and-a-halfmillionsimulatedquantificationswithvarioususer-definedconditions.Comparedwithclassical approaches,wefoundthatRIMS-baseddrqPCRprovidedsuperiorprecisionandcomparableaccuracy. Conclusions/Significance: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy numbervariationsetc.Also,lab-to-labcomparabilitycanbegreatlysimplified. Citation: Bernth Jensen JM, Petersen MS, Stegger M, ?stergaard LJ, M?ller BK (2010) Real-Time Relative qPCR without Reference to Control Samples and EstimationofRun-SpecificPCRParametersfromRun-InternalMini-StandardCurves.PLoSONE5(7):e11723.doi:10.1371/journal.pone.0011723 Editor:M.ThomasP.Gilbert,NaturalHistoryMuseumofDenmark,Denmark ReceivedFebruary15,2010;AcceptedJune2