Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR.
Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR.
复制标题
DOI：
10.1186/1472-6750-7-21
复制
发表时间：
2007-05-04
影响因子：
3.5
通讯作者：
Baldwin KM
中科院分区：
文献类型：
--
作者：
Haddad F;Qin AX;Giger JM;Guo H;Baldwin KM
准确测量基因表达模式的能力在研究基因功能中是必不可少的。逆转录聚合酶链反应（RT-PCR）已成为检测和测量细胞和少量组织中RNA表达模式的首选方法。我们之前的研究结果表明，使用一种流行的RNase H- RT酶可以显著地合成与引物无关的cDNA。在不添加RT引物的情况下，从RT反应中扩增出PCR产物。这一发现危及RT-PCR在分析两个方向表达的RNA时的准确性。目前的文献发现表明，自然发生的反义表达在哺乳动物转录组中广泛存在，并由编码和非编码调节RNA组成。本研究的主要目的是研究不依赖于引物的cDNA合成的发生，以及它如何影响正反义RNA对检测的准确性。我们对细胞RNA和体外合成RNA的研究结果表明，这些产物可能是RNA自引产生随机cDNA产物的结果，这导致了链特异性的丧失。使用RNase H+ RT酶，在高温（50℃）下进行RT反应，大大提高了RT- pcr检测的链特异性。虽然RT PCR是检测和定量细胞中RNA表达的基本方法，但不依赖引物的cDNA合成会干扰RT的特异性，并可能导致结果的误解，尤其是在同时表达正义和反义RNA的情况下。为了准确地解释结果，必须进行适当的阴性对照。
The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs. Our findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection. While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.
登录
查看更多内容
影响因子：
64.5
作者：
Cawley, S;Bekiranov, S;Gingeras, TR
通讯作者：
Gingeras, TR
影响因子：
2.7
作者：
GUNJI, T;KATO, N;SHIMOTOHNO, K
通讯作者：
SHIMOTOHNO, K
影响因子：
3.5
作者：
Seitz, A;Gourevitch, D;Heber-Katz, E
通讯作者：
Heber-Katz, E
影响因子：
7
作者：
Dahary, D;Elroy-Stein, O;Sorek, R
通讯作者：
Sorek, R
DOI：
10.1152/ajpheart.01111.2005
发表时间：
2006-06-01
影响因子：
4.8
作者：
Haddad, F;Qin, AX;Baldwin, KM
通讯作者：
Baldwin, KM