Bookout Angie L, Cummins Carolyn L, Mangelsdorf David J, Pesola Jean M, Kramer Martha F
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Curr Protoc Mol Biol. 2006 Feb;Chapter 15:Unit 15.8. doi: 10.1002/0471142727.mb1508s73.
Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.
本单元提供了关于实时定量聚合酶链反应（QPCR）用于基因表达分析的详细内容。这些方案是为高通量384孔格式仪器（如应用生物系统公司的7900HT）设计的，但可进行修改以适用于任何实时PCR仪器。文中讨论了QPCR引物和探针的设计及验证，并描述了三种相对定量方法：标准曲线法、效率校正DeltaCt法和比较循环时间法（即DeltaDeltaCt法）。此外，还提供了一种对未知样品中的RNA进行绝对定量的方法。RNA标准品与实验样品以相同方式进行逆转录-聚合酶链反应（RT-PCR），从而兼顾了两个过程的反应效率。本方案描述了用于实时和非实时RT-PCR应用的合成RNA分子的制备和定量。
