Bio-Rad Laboratories Canada Inc., 1329 Meyerside Drive, Mississauga, Ontario L5T1C9, Canada.
Bio-Rad Laboratories Canada Inc., 1329 Meyerside Drive, Mississauga, Ontario L5T1C9, Canada.
Trends Biotechnol. 2019 Jul;37(7):761-774. doi: 10.1016/j.tibtech.2018.12.002. Epub 2019 Jan 14.
Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
定量聚合酶链式反应 (qPCR) 是生物和环境样本中核酸分子定量的最常用技术之一。尽管该方法被认为相对简单，但有许多步骤和试剂需要优化和验证，以确保数据具有可重复性，准确反映所提出的生物学问题。本文描述并说明了 qPCR 实验中的关键缺陷和误差源，并提供了一个严格的、逐步的流程，每次都可最大限度地减少产生可重复、有发表价值数据的变异性、时间和成本。最后，还介绍了在 qPCR 和数字 PCR 技术之间做出明智选择的方法。
