Sato M, Mizobuchi M, Murao K, Tamaki M, Takahara J
Department of Medicine, Kagawa Medical School.
J Biochem. 1994 Dec;116(6):1198-201. doi: 10.1093/oxfordjournals.jbchem.a124662.
A simple and efficient method for preparing rcRNA through competitive RT-PCR has been developed. The basis of this method is the use of a false-priming PCR product including the same primers as a specific product. A 290 bp fragment obtained by two-step PCR was subcloned into a plasmid vector and then the cloned DNA was transcribed into rcRNA. After competitive RT-PCR using sample RNA and rcRNA had been carried out, Southern blot hybridization was performed. The method was applied to determine the amounts of PIMT mRNAs in the rat pituitary. The quantitative analysis indicated that an at least 2-fold difference in PIMT mRNA level can be accurately determined with our method.
已经开发出一种通过竞争性逆转录聚合酶链反应（RT-PCR）制备rcRNA的简单有效方法。该方法的基础是使用一种假引发PCR产物，其包含与特定产物相同的引物。通过两步PCR获得的290 bp片段被亚克隆到质粒载体中，然后将克隆的DNA转录为rcRNA。在用样品RNA和rcRNA进行竞争性RT-PCR后，进行Southern印迹杂交。该方法用于测定大鼠垂体中PIMT mRNA的含量。定量分析表明，用我们的方法可以准确测定PIMT mRNA水平至少2倍的差异。
