Mejora Genética Vegetal, Instituto de Agricultura Sostenible, CSIC, 14080 Córdoba, Spain.
J Exp Bot. 2012 Oct;63(17):6069-77. doi: 10.1093/jxb/ers276. Epub 2012 Oct 8.
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is probably the most common molecular technique used in transcriptome analyses today. The simplicity of the technology and associated protocols that generate results without the need to understand the underlying principles has made RT-qPCR the method of choice for RNA quantification. Rather than the 'gold standard technology' often used to describe it, the performance of RT-qPCR suffers from considerable pitfalls during general workflow. The inconsistency of conventional methods for the evaluation of RNA quality and its influence on qPCR performance as well as stability of reference genes is summarized and discussed here.
逆转录-定量实时聚合酶链反应 (RT-qPCR) 可能是当今转录组分析中最常用的分子技术。该技术的简单性及其相关协议使得结果无需理解潜在原理即可生成，这使得 RT-qPCR 成为 RNA 定量的首选方法。RT-qPCR 的性能在一般工作流程中存在相当多的缺陷，而不是经常用来描述它的“黄金标准技术”。本文总结和讨论了评估 RNA 质量的常规方法的不一致性及其对 qPCR 性能以及参照基因稳定性的影响。
