High-throughput real-time quantitative reverse transcription PCR.
High-throughput real-time quantitative reverse transcription PCR.
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DOI：
10.1002/0471142727.mb1508s73
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发表时间：
2006-02-01
影响因子：
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通讯作者：
Kramer, Martha F
中科院分区：
文献类型：
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作者：
Bookout, Angie L;Cummins, Carolyn L;Kramer, Martha F
本单元提供了实时定量聚合酶链反应（QPCR）用于基因表达分析的广泛细节。该方案是为高通量、384孔格式的仪器（如Applied Biosystems 7900HT）设计的，但也可以修改以适应任何实时PCR仪器。讨论了QPCR引物和探针的设计和验证，并描述了三种相关的定量方法：标准曲线法、效率修正的DeltaCt法和比较周期时间（DeltaDeltaCt法）。此外，还提供了未知样品中RNA的绝对定量方法。RNA标准品以与实验样品相同的方式进行RT-PCR，从而考虑到两种程序的反应效率。本协议描述了用于实时和非实时RT-PCR应用的合成RNA分子的生产和定量。
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.