Ashton Kevin John, Headrick John Patrick
Heart Foundation Research Centre, Griffith University, Southport, Australia.
Methods Mol Biol. 2007;366:121-43. doi: 10.1007/978-1-59745-030-0_7.
Quantitative (real-time) PCR (qPCR) represents a highly sensitive, sequence-specific, and reproducible technique for the gel-free detection and quantitation of nucleic acids. Owing to its large dynamic range and throughput, this approach has become the chosen method for rapid quantification of mRNA levels in biological samples. The sensitivity of this method permits the reliable detection of low concentrations of initial template, while delivering a linear range of up to 10 orders of magnitude in copy number. This chapter details the basic methodology behind key components of a qPCR experiment, including sample preparation, fluorescent chemistries, primer/probe design, and data analysis applicable to cardiovascular research.
定量（实时）PCR（qPCR）是一种用于无凝胶检测和定量核酸的高灵敏度、序列特异性且可重复的技术。由于其较大的动态范围和通量，这种方法已成为快速定量生物样品中mRNA水平的首选方法。该方法的灵敏度允许可靠地检测低浓度的初始模板，同时在拷贝数上提供高达10个数量级的线性范围。本章详细介绍了qPCR实验关键组件背后的基本方法，包括样品制备、荧光化学、引物/探针设计以及适用于心血管研究的数据分析。
