Quantification of mRNA using real-time RT-PCR
Quantification of mRNA using real-time RT-PCR
复制标题
DOI：
10.1038/nprot.2006.236
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Bustin, Stephen A.
中科院分区：
文献类型：
--
作者：
Nolan, Tania;Hands, Rebecca E.;Bustin, Stephen A.
实时逆转录聚合酶链式反应(RT-qPCR)解决了分子医学、生物技术、微生物学和诊断学对定量数据分析的明显需求，已成为定量检测mRNA的首选方法。尽管它经常被描述为“黄金”标准，但它远不是一个标准的化验方法。RNA模板、分析设计和方案的可变性以及不适当的数据标准化和不一致的数据分析所造成的重大问题是众所周知的，但也被广泛忽视。作为迈向标准化的第一步，我们描述了一系列RT-qPCR协议，这些协议说明了生成可靠和可重复性的定量数据所需的基本技术步骤。然而，我们想强调的是，RT-qPCR数据仅构成关于细胞或组织中给定转录本数量的信息的快照。对可变信使核糖核酸水平的生物学影响的任何评估都必须包括有关调节RNA、蛋白质水平和蛋白质活性的额外信息。这里描述的整个方案，包括从最初的分析设计到可靠的qPCR数据分析的所有阶段，大约需要15小时。
The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.