ABSOLUTE MESSENGER-RNA QUANTIFICATION USING THE POLYMERASE CHAIN-REACTION (PCR) - A NOVEL-APPROACH BY A PCR AIDED TRANSCRIPT TITRATION ASSAY (PATTY)
ABSOLUTE MESSENGER-RNA QUANTIFICATION USING THE POLYMERASE CHAIN-REACTION (PCR) - A NOVEL-APPROACH BY A PCR AIDED TRANSCRIPT TITRATION ASSAY (PATTY)
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DOI：
10.1093/nar/17.22.9437
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发表时间：
1989-11-25
影响因子：
14.9
通讯作者：
HAHLBROCK, K
中科院分区：
文献类型：
--
作者：
BECKERANDRE, M;HAHLBROCK, K
聚合酶链反应（PCR）被用作mRNA绝对定量的新方法的一部分。我们描述了一种PCR辅助的转录物滴定分析（PATTY），它是基于体外产生的转录物的共扩增，该转录物与靶mRNA的单碱基交换不同。将总RNA样品的相同部分“掺入”不同量的该突变的标准RNA，转化为cDNA并通过PCR扩增。因为碱基交换产生了新的限制性内切酶位点，所以可以在限制性内切酶消化和通过凝胶电泳分离之后确定源自靶mRNA的共扩增DNA与源自标准RNA的扩增DNA的比率。该方法在24小时内给出准确的结果，特别适用于定量存在于非常少量总RNA中的低丰度mRNA或更丰富的mRNA。马铃薯（Solanum tuberosum L.）是在一个案例研究中测量的。新方法可以准确地检测每个分析约100个分子。
The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amount of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance encoding 4-coumarate:CoA ligase (4CL) in cultured potato cell (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.