Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems
Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems
复制标题
DOI：
10.1677/jme.0.0290023
复制
发表时间：
2002-08-01
影响因子：
3.5
通讯作者：
Bustin, SA
中科院分区：
文献类型：
--
作者：
Bustin, SA
基于荧光的实时逆转录聚合酶链式反应(RT-PCR)被广泛应用于稳态mRNA水平的定量，是基础研究、分子医学和生物技术的重要工具。检测方法易于操作，能够高通量，并且可以结合高灵敏度和可靠的特异度。然而，随着新的酶、化学和仪器的引入，这项技术正在迅速发展，尽管实时RT-PCR解决了传统RT-PCR固有的许多困难，但越来越明显的是，它产生了需要迫切关注的新问题。因此，除了提供实时RT-PCR最新技术的快照之外，这篇综述还有一个额外的目的：它将描述和批判性地讨论与解释结果相关的一些问题，这些结果是数字的，有助于统计分析，但其准确性受到试剂和操作员变异性的显著影响。
The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation, However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.