Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis
Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis
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DOI：
10.2144/98251rr01
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发表时间：
1998-07-01
期刊：
影响因子：
2.7
通讯作者：
Landers, JP
中科院分区：
文献类型：
--
作者：
Borson, ND;Strausbauch, MA;Landers, JP
人们经常尝试对 mRNA 进行半定量，以此作为避免使用竞争对手模板所固有的费力且耗时的定量过程的一种手段。然而，半定量方法存在产生不可重复的数据骰子的风险，从而导致管与管之间的变异性和/或对 PCR 平台期产生的产物数量的误解。随后，很难比较来自单独实验的半定量数据，并且无法比较用不同引物对扩增的基因的mRNA转录物水平。因此，可靠的 mRNA 定量方法仍然依赖于内部标准化的使用。在本报告中，我们描述了一种基于定量竞争性逆转录 PCR (QC-RT-PCR) 与毛细管电泳 (CE) 相结合的低丰度 mRNA 转录本的可靠定量策略，以实现产物的快速分离和缺陷。其中包括关于 RNA 竞争剂设计的建议，这些竞争剂可以与靶 RNA 配对，用于用基因特异性引物引发的 cDNA 合成；然后使用单个引物对在同一管中直接对这些合成的 cDNA 进行共扩增。我们描述了（i）单管 RT-PCR 方案，该方案在每个关键酶促步骤的相同反应环境中提供 cDNA 合成和随后的靶标和竞争物 PCR 扩增，（ii）用于优化精确和一致 PCR 扩增的独特热启动规定，以及（iii）通过 CE 和激光诱导荧光快速分离、检测和定量 PCR 产物的方法。
Attempts are frequently made to semi-quantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However semiquantitative approaches present the risk of generating non-reproducible data dice to tube-to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semi-quantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and defection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single-tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.