A comparison of methods for RNA extraction from lymphocytes for RT-PCR.
A comparison of methods for RNA extraction from lymphocytes for RT-PCR.
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RT-PCR 中淋巴细胞 RNA 提取方法的比较。
DOI：
10.1101/gr.4.3.185
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Raine,CS
中科院分区：
文献类型：
--
作者：
Liedtke,W;Battistini,L;Brosnan,CF;Raine,CS
已经描述了从非贴壁哺乳动物细胞中提取RNA的各种方法，或者在伴随各种市售试剂的方案中提供了各种方法。(1-1~逆转录酶-PCR（RT-PCR）允许扩增，甚至定量，以前检测不到的mRNA量。(11（1）所引用方法的比较（考虑到RT-PCR的重要性，cDNA转录物的有限稀释测定是比其它RNA参数如分光光度法、北方印迹等更好的终点）; (2)总RNA或多聚（A）RNA提取是否更有利于随后的RT-PCR;和（3）用oligo（dT）、随机六聚体或RT-PCR下游引物的cDNA合成的详细比较。我们通过从人类淋巴细胞中提取RNA解决了这些问题。逆转录提取的RNA [poly（A）或总RNA]，并对cDNA进行PCR扩增[β-肌动蛋白，CD 3，和//BTCR重排VB 2]。（~ 3-15~
Various methods have been described to extract RNA from nonadherent mammalian cells or are provided in protocols accompanying various commercially available reagents.(1-1~ Reverse transcriptase-PCR (RT-PCR) allows the amplification, even the quantitation, of previously undetectable amounts of mRNA.(11'1z) In this burgeoning field, some major points of interest have not yet been investigated:(1) comparison of the cited methods (given the importance of RT-PCR, limiting dilutional assays of cDNA transcripts is a better end point than other RNA parameters like spectrophotometry, Northern blotting, etc.);(2) whether total RNA or poly (A) RNA extraction is more favorable for subsequent RT-PCR; and (3) a detailed comparison of cDNA synthesis with oligo (dT), random hexamer, or RT-PCR downstream primers. We have addressed these questions by extracting RNA from human lymphocytes. The extracted RNA [either poly (A) or total] was reverse-transcribed, and the cDNA was subjected to PCR-amplifying [3-actin, CD3, and~//BTCR rearrangement VB2J~ l.(~ 3-15~