Peirson Stuart N, Butler Jason N
Division of Circadian and Visual Neuroscience, Univesity of Oxford, UK.
Methods Mol Biol. 2007;362:349-62. doi: 10.1007/978-1-59745-257-1_25.
Quantitative PCR (qPCR) has entered widespread use with the increasing availability of real-time PCR. By the incorporation of fluorescent dyes in the reaction mixture, increases in amplification products can be monitored throughout the reaction, enabling measurements to be taken in the exponential phase of the reaction, before the reaction plateau. Whatever the platform or chemistry involved, the starting point of a real-time assay is a tissue-specific RNA and the end point of a real-time reaction is an amplification plot. As such, rather than focusing on specific platforms or chemistries, herein we address the basic principles that underlie sample preparation, experimental design, use of internal controls, assay considerations, and approaches to data analysis. The advent of real-time PCR has enabled high-throughput analysis of multiple transcripts from small tissue samples, with an unparalleled dynamic range and sensitivity. However, to new users, this technique may seem to require extensive optimization and troubleshooting to obtain reliable data; this is further compounded by the mass of technical variations present throughout the literature. The aim of this article is to provide the necessary basics to get a quantitative real-time PCR assay up and running, and to address some of the problems that may arise and how these may be resolved.
随着实时荧光定量PCR（qPCR）技术的日益普及，定量PCR已得到广泛应用。通过在反应混合物中加入荧光染料，可以在整个反应过程中监测扩增产物的增加情况，从而能够在反应达到平台期之前的指数期进行测量。无论涉及何种平台或化学方法，实时检测的起点都是组织特异性RNA，终点是扩增曲线。因此，本文并非专注于特定的平台或化学方法，而是阐述样本制备、实验设计、内参使用、检测注意事项以及数据分析方法等方面的基本原理。实时荧光定量PCR的出现使得从小组织样本中对多个转录本进行高通量分析成为可能，其动态范围和灵敏度无与伦比。然而，对于新用户来说，这项技术似乎需要进行大量的优化和故障排除才能获得可靠的数据；而文献中大量的技术差异更是加剧了这一问题。本文旨在提供必要的基础知识，以使定量实时荧光定量PCR检测能够顺利开展，并解决可能出现的一些问题以及如何解决这些问题。
