Nolan Tania, Hands Rebecca E, Bustin Stephen A
Sigma-Aldrich, Homefield Road, Haverhill, UK.
Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.
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.
实时逆转录聚合酶链反应（RT-qPCR）满足了分子医学、生物技术、微生物学和诊断领域对定量数据分析的明显需求，已成为mRNA定量的首选方法。尽管它常被描述为“金”标准，但远非标准检测方法。RNA模板的变异性、检测设计和方案以及不适当的数据归一化和不一致的数据分析所导致的重大问题广为人知，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方案，阐述了生成可靠且可重复的定量数据所需的基本技术步骤。然而，我们要强调的是，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快照。对可变mRNA水平的生物学后果的任何评估都必须包括有关调节性RNA、蛋白质水平和蛋白质活性的额外信息。此处描述的整个方案，涵盖从初始检测设计到可靠的qPCR数据分析的所有阶段，大约需要15小时。
