Wong Marisa L, Medrano Juan F
Department of Animal Science, University of California, Davis 95616-8521, USA.
Biotechniques. 2005 Jul;39(1):75-85. doi: 10.2144/05391RV01.
Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.
实时荧光定量PCR已成为应用最广泛的基因定量方法之一，因为它具有较宽的动态范围、极高的灵敏度、高度的序列特异性、极少或无需扩增后处理，并且易于提高样品通量。然而，要充分利用这些优势，需要清楚了解进行实时荧光定量PCR实验的众多可用选项。本文从实时荧光定量PCR背后的理论入手，讨论了实时荧光定量PCR实验的关键组成部分，包括一步法或两步法PCR、绝对定量与相对定量、可用于相对定量和扩增效率计算的数学模型、归一化或数据校正的类型以及检测化学方法。此外，还讨论了变异的多种原因以及计算批内和批间变异的方法。
