Yeatts Kimberly
Diabetes, Cardiovascular, and Metabolic Diseases Division, Translational Genomics Research Institute, Phoenix, AZ, USA.
Methods Mol Biol. 2011;700:171-84. doi: 10.1007/978-1-61737-954-3_12.
Quantitative PCR (qPCR) is considered the gold standard for molecular DNA quantification and can be used for a wide range of techniques from comparing gene expression levels to quantifying DNA copy number variation. The strengths of this assay include sensitivity to a wide range of expression levels, low starting template requirement, which is important when samples are scarce, and quick turnaround time. However, there are many variables to consider when performing qPCR: including (1) starting materials (e.g., tissues, cells, or genomic DNA), (2) fluorescent detection (e.g., SYBR green dye, fluorescent probes, or multiplexed assays), and (3) analysis methods (e.g., simple equations with single reference genes or complex algorithms with multiple reference genes). This chapter will introduce the process of designing an experiment while avoiding common mistakes and present tools for performing qPCR in a practical, simple, and efficient manner.
定量聚合酶链反应（qPCR）被认为是分子DNA定量的金标准，可用于从比较基因表达水平到定量DNA拷贝数变异等广泛的技术。该检测方法的优点包括对广泛表达水平的敏感性、低起始模板要求（当样本稀缺时这一点很重要）以及快速周转时间。然而，进行qPCR时需要考虑许多变量：包括（1）起始材料（例如组织、细胞或基因组DNA）、（2）荧光检测（例如SYBR绿染料、荧光探针或多重检测）以及（3）分析方法（例如使用单个参考基因的简单方程或使用多个参考基因的复杂算法）。本章将介绍实验设计过程，同时避免常见错误，并介绍以实用、简单和高效的方式进行qPCR的工具。
