Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Methods. 2010 Apr;50(4):227-30. doi: 10.1016/j.ymeth.2009.11.001. Epub 2009 Dec 5.
Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting.
逆转录定量 PCR（RT-qPCR）被认为是准确、敏感和快速测量基因表达的金标准。然而，许多用户没有意识到的是，在得出有生物学意义和可信赖的结论之前，需要解决工作流程中的许多关键问题。在这里，我们回顾了从规划和准备阶段，到实际实时 PCR 循环实验，再到数据分析和报告步骤的整个工作流程。这个过程可以用适当的缩写 PCR 来表示：计划/准备、循环和报告。关键信息是，从活细胞到核酸提取、储存、各种酶处理步骤（如 DNA 酶处理、逆转录和 PCR 扩增），再到数据分析，最后是报告，质量保证和质量控制在整个 RT-qPCR 工作流程中都是必不可少的。
