VanGuilder Heather D, Vrana Kent E, Freeman Willard M
Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.
Biotechniques. 2008 Apr;44(5):619-26. doi: 10.2144/000112776.
Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.
25年前发明后，聚合酶链反应（PCR）已被应用于众多分子生物学领域。逆转录定量PCR（RT-qPCR）进行基因表达分析一直是后基因组时代的一项关键支撑技术。自《生物技术》创刊以来，本杂志一直是qPCR技术改进、实验设计和数据分析的资源。qPCR，更具体地说是实时qPCR，已成为测量目标基因表达、验证微阵列实验和监测生物标志物的常规且可靠的方法。实时qPCR的使用几乎已取代了其他方法（如Northern印迹法、核糖核酸酶保护分析）。鉴于该方法已发展到成熟的开发和应用阶段，本综述探讨了用于基因表达分析的qPCR的当前状况。具体而言，讨论了实时qPCR的不同荧光报告技术以及内参的选择。还介绍了数据分析方法的概念框架，以揭开这些分析技术的神秘面纱。随着该技术变得更快、更具成本效益、更易于使用且能够实现更高通量，qPCR的未来依然光明。
