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      A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.

      1 ,
      Analytical biochemistry
      Elsevier BV

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          Abstract

          Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative C(T) method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative C(T) method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.

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          Author and article information

          Journal
          Anal Biochem
          Analytical biochemistry
          Elsevier BV
          0003-2697
          0003-2697
          Mar 01 2002
          : 302
          : 1
          Affiliations
          [1 ] Department of Physiology, University of Adelaide, Adelaide, SA, 5005, Australia.
          Article
          S0003269701955307
          10.1006/abio.2001.5530
          11846375
          95b78d7b-6fd3-4e46-a1f6-510eae0b646a
          History

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