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DA: A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 2002
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@MISC{Liu_da:a,
author = {Weihong Liu and David A. Saint},
title = {DA: A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 2002},
year = {}
}
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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 CT 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 CT 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. © 2002 Elsevier Science (USA) The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of specific mRNAs (1, 2). In contrast with classic techniques, such as Northern blot analysis and RNase protection assays, which require large amounts of total
Keyphrases
anal biochem polymerase chain reaction kinetics new quantitative method amplification efficiency new method standard curve method comparative ct method sensitive method gene transcript gene expression level quantitative method standard curve kinetic process relative level individual reaction validation experiment relative standard curve method kinetic curve rnase protection assay two-pore potassium channel elsevier science gene expression accurate approach low-abundance mrna classic technique real-time pcr specific mrna reverse transcription polymerase chain reaction real-time reverse transcription reference gene routine rt-pcr northern blot analysis relative quantitative method initial amount large amount target gene
