PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency.
PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency.
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DOI：
10.1093/nar/gni176
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发表时间：
2005-11-27
影响因子：
14.9
通讯作者：
Steindler DA
中科院分区：
文献类型：
--
作者：
Suslov O;Steindler DA
本研究解决了逆转录酶抑制 PCR 的问题。研究表明，这种抑制主要发生在取少量RNA进行RT反应时，对于很少表达的转录本更明显。我们在此表明，无论 RT 使用多少模板，都会发生抑制。抑制具有全局性，即任何给定转录物的扩增可能会受到不同水平的抑制的影响。当使用未纯化 RT 样品的连续稀释来构建校准曲线时，抑制过程还解释了错误得出的 PCR 扩增效率，有时超过 100%。 RT 不仅通过抑制 PCR 来影响 PCR。当取微克 RNA 进行 RT 反应时，逆转录酶可能会在某些 PCR 条件下导致某些转录物过度扩增。提出了RT对PCR影响的可能机制，并采用纯化方法消除RT对PCR的影响。
This study addresses the problem of PCR inhibition by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA is taken for RT reaction, and it is more visible for rarely expressed transcripts. We show here that the inhibition takes place regardless of what amount of template is utilized for RT. The inhibition possesses a global nature, i.e. the amplification of any given transcript may be compromised with different levels of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, sometimes more than 100%, when the sequential dilutions of unpurified RT sample are utilized to build the calibration curve. The RT influences PCR not only by inhibiting it. When microgram(s) of RNA are taken for RT reaction, reverse transcriptase may cause overamplification of some transcripts under certain PCR conditions. The possible mechanism of RT influence on PCR is presented, and a purification method is implemented to remove the effects of RT on PCR.
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影响因子：
14.9
作者：
Liss, B
通讯作者：
Liss, B
DOI：
10.1073/pnas.77.3.1316
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
MYERS, JC;DOBKIN, C;SPIEGELMAN, S
通讯作者：
SPIEGELMAN, S
影响因子：
2.9
作者：
Nycz, CM;Dean, CH;Walker, GT
通讯作者：
Walker, GT
影响因子：
14.9
作者：
SELLNER, LN;COELEN, RJ;MACKENZIE, JS
通讯作者：
MACKENZIE, JS
影响因子：
4.8
作者：
Schultz, SJ;Zhang, MH;Champoux, JJ
通讯作者：
Champoux, JJ