Pitfalls of reverse transcription quantitative polymerase chain reaction standardization: Volume-related inhibitors of reverse transcription

被引:11
作者
Pugniere, Pascal [1 ]
Banzet, Sebastien
Chaillou, Thomas
Mouret, Catherine [1 ]
Peinnequin, Andre [1 ]
机构
[1] IRBA La Tronche, Genom Core Facil, F-38702 La Tronche, France
关键词
Reverse transcription; RT-qPCR; mRNA quantification; Standardization; Inhibitor; MIQE guidelines; MESSENGER-RNA; REAL-TIME; SECONDARY STRUCTURE; GENE-EXPRESSION; PCR; QUANTIFICATION; EXTRACTION;
D O I
10.1016/j.ab.2011.04.008
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RI step (CA-RI). Because RNA isolation yields are variable, such a practice requires the use of variable volumes of nucleic acid extracts in RI reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decrease in RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have a slight effect on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RI). We show that CV-RT, compared with the usual CA-RI, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification. (C) 2011 Elsevier Inc. All rights reserved.
引用
收藏
页码:151 / 157
页数:7
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