Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR.
Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR.
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几种商业逆转录酶的检测极限：对通过定量RT-PCR评估的低丰度转录水平的影响。
DOI：
10.1186/1471-2199-8-93
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发表时间：
2007-10-22
影响因子：
--
通讯作者：
Bissonnette N
中科院分区：
文献类型：
--
作者：
Levesque-Sergerie JP;Duquette M;Thibault C;Delbecchi L;Bissonnette N
在功能基因组学中，转录本的测量是至关重要的。定量逆转录聚合酶链反应（qRT-PCR）测定是最流行的技术，并且依赖于初始分子步骤，即逆转录（RT）。这项研究提供了一个复杂的概述，如RT系统，背景RNA的量和转录本丰度的qRT-PCR的效率的影响。采用qRT-PCR技术，比较了几种常用的RT系统的效率，测定了PCR扩增产物的产量以及PCR抑制剂含量的影响。使用TaqMan系统进行qRT-PCR测定，尽管我们还测试了SYBR绿色I化学，其与所有RT系统不兼容。当处理低丰度转录物时，SuperScript II系统产生的可检测分子比测试的其他四个系统：Sensiscript，Omniscript，SuperScript III和PowerScript更多（P < 0.05）。然而，在1至2 μg背景RNA存在下，Sensiscript和PowerScript系统对于检测高丰度转录本更有效（P < 0.05）。最引人注目的方面是RT反应的稀释对随后的PCR的影响。事实上，当稀释的RT反应用于定量PCR测量时，释放了一些抑制。此外，RT反应中背景RNA的量也是影响qRT-PCR下游步骤（PCR反应）的主要因素。尽管Sensiscript的偏倚较小，但其他系统含有PCR抑制剂的重要来源，对qRT-PCR的干扰高达70%。本研究提供了RT系统、qRTPCR化学、背景RNA量和转录本丰度等因素对qRT-PCR效率的影响的综合概述。尽管最显著的影响因素是RT系统中抑制剂的存在，但总背景RNA也是影响PCR反应的主要影响组分。当研究的目的是获得精确的基因表达测量或分析全局转录组（例如微阵列）时，RT步骤至关重要，应仔细检查。
In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT). This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P < 0.05). However, the Sensiscript and PowerScript systems were more efficient for detecting high-abundance transcripts in the presence of 1 to 2 μg background RNA (P < 0.05). The most striking aspect was the influence of the dilution of the RT reaction on the subsequent PCR. Indeed, some inhibition was released when diluted RT reactions were used for the quantitative PCR measurements. Furthermore, the amount of background RNA in the RT reaction was also a major component influencing a downstream step in qRT-PCR, the PCR reaction. Whereas Sensiscript was less biased, the other systems contained an important source of PCR inhibitors, interfering as much as 70% with the qRT-PCR. This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray), the RT step is critical and should be examined with care.
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影响因子：
14.9
作者：
Suslov O;Steindler DA
通讯作者：
Steindler DA
影响因子：
2.9
作者：
Nycz, CM;Dean, CH;Walker, GT
通讯作者：
Walker, GT
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
DOI：
10.1093/bfgp/2.1.31
发表时间：
2003-04-01
期刊：
Briefings in Functional Genomics & Proteomics
影响因子：
--
作者：
Livesey, F. J.
通讯作者：
Livesey, F. J.
影响因子：
4.4
作者：
Cao W;Epstein C;Liu H;DeLoughery C;Ge N;Lin J;Diao R;Cao H;Long F;Zhang X;Chen Y;Wright PS;Busch S;Wenck M;Wong K;Saltzman AG;Tang Z;Liu L;Zilberstein A
通讯作者：
Zilberstein A