A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction
A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction
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DOI：
10.1006/mcpr.2002.0405
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发表时间：
2002-04-01
影响因子：
3.3
通讯作者：
Ko, Y
中科院分区：
文献类型：
--
作者：
Fronhoffs, S;Totzke, G;Ko, Y
核酸的定量，尤其是信使核糖核酸的定量，在生物医学研究中越来越重要。近年来发展起来的实时定量聚合酶链式反应(PCR)是一种快速、准确、重复性好的高灵敏度基因表达定量技术，与传统的定量聚合酶链式反应相比具有明显的优势。通过外推来自代表给定荧光信号的初始拷贝数的标准校准曲线的荧光信号，在PCR反应的指数阶段执行转录本定量。我们开发了一种基于LightCycler(TM)辅助的实时荧光聚合酶链式反应的基因转录定量方法，并结合一种简单快速的方法来构建与靶基因相同的基因序列的外部cRNA标准品。用T7RNA聚合酶体外转录、逆转录、实时定量聚合酶链式反应合成CRNAs。我们应用这种方法对正常睾丸组织中真核细胞起始因子3p110(EIF3S8)的mRNA进行了转录定量。我们还提出了一种快速而简单的策略来构建用于实时聚合酶链式反应的cRNA标准。(C)2002年爱思唯尔科学有限公司。
Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCycler(TM) - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR. (C) 2002 Elsevier Science Ltd.