Anderson K M, Cheung P H, Kelly M D
Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Karen M
J Pharmacol Toxicol Methods. 1997 Nov;38(3):133-40. doi: 10.1016/s1056-8719(97)00078-6.
Sensitive and quantitative measurement of messenger RNA (mRNA) is important for accurate assessment of gene expression. Conventional methods of mRNA measurement frequently lack the sensitivity required to detect mRNA expressed at low level, such as mRNA encoding receptors and intracellar signaling molecules. Thus, the extremely sensitive RT-PCR has become the method of choice for examination of gene expression. However, quantitation of mRNA by PCR is difficult because small variations in amplification efficiencies among sample tubes can lead to substantial differences in product yield, thereby rendering direct comparisons between samples invalid. Development of protocols for quantitative RT-PCR has relied on internal standards to monitor the efficiency of the RT-PCR in different reaction tubes. Technically, the two most serious limitations to routine successful application of competitive quantitative PCR is ready access to competitive internal standards and efficient methods for accurate quantitative analysis of the data. In the present manuscript, application and validation of a simple approach to generate homologous internal competitive standards and to quantitate data for rapid, accurate determination of the expression level of genes by quantitative PCR is described. Generation of the competitive standard from a previously amplified PCR product by the methods described requires only one additional primer pair, and an additional two-step reaction; it can be completed in 1-2 days. Analyzing the results of the competitive PCR reaction via phosphoimager analysis provides a simple, rapid method for accurate quantitation of results. Data presented here clearly illustrate that the methods described have been successfully applied, and that they should have wide application for competitive quantitative PCR analysis of gene expression.
信使核糖核酸（mRNA）的灵敏且定量检测对于准确评估基因表达非常重要。传统的mRNA检测方法常常缺乏检测低水平表达的mRNA（如编码受体和细胞内信号分子的mRNA）所需的灵敏度。因此，极其灵敏的逆转录聚合酶链反应（RT-PCR）已成为检测基因表达的首选方法。然而，通过PCR对mRNA进行定量很困难，因为样品管之间扩增效率的微小差异会导致产物产量出现显著差异，从而使样品之间的直接比较无效。定量RT-PCR方案的开发依赖于内标来监测不同反应管中RT-PCR的效率。从技术上讲，常规成功应用竞争性定量PCR的两个最严重限制是难以获得竞争性内标以及缺乏准确分析数据的有效方法。在本论文中，描述了一种简单方法的应用和验证，该方法可生成同源内部竞争标准品并对数据进行定量，以便通过定量PCR快速、准确地测定基因表达水平。通过所述方法从先前扩增的PCR产物生成竞争标准品仅需要一对额外的引物和一个额外的两步反应；整个过程可在1至2天内完成。通过磷光成像分析来分析竞争性PCR反应的结果，为准确定量结果提供了一种简单、快速的方法。此处呈现的数据清楚地表明所述方法已成功应用，并且它们在基因表达的竞争性定量PCR分析中应具有广泛的应用。
