Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/11717
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Type: Journal article
Title: A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics
Author: Liu, W.
Saint, D.
Citation: Analytical Biochemistry, 2002; 302(1):52-59
Publisher: Academic Press Inc
Issue Date: 2002
ISSN: 0003-2697
1096-0309
Statement of
Responsibility: 
Weihong Liu and David A. Saint
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.
Keywords: Animals; Mice; Rats; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Kinetics
Description: © 2007 Elsevier B.V. All rights reserved.
RMID: 0020020034
DOI: 10.1006/abio.2001.5530
Description (link): http://www.elsevier.com/wps/find/journaldescription.cws_home/622781/description#description
Appears in Collections:Physiology publications

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