A multi-species assay for siRNA-mediated mRNA knockdown analysis without the need for RNA purification

被引：2

作者：

Wang, Zhibin ^{[1
]}

Pacchione, Stephen J. ^{[1
]}

Niu, Zhutian ^{[1
]}

Troilo, Philip J. ^{[1
]}

Griffiths, Thomas G. ^{[1
]}

Striano, Krista L. ^{[1
]}

Lebron, Jose A. ^{[1
]}

Wolf, Jayanthi J. ^{[1
]}

机构：

[1] Merck & Co Inc, Merck Res Labs, Dept Safety Assessment, West Point, PA 19486 USA

来源：

JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS | 2011年 / 63卷 / 02期

关键词：

mRNA quantification method; Stem-loop RT-qPCR; siRNA-mediated mRNA knockdown; REAL-TIME PCR; APOLIPOPROTEIN-B; GENE; CHOLESTEROL; QUANTIFICATION; QUANTITATION; PROTEIN; TISSUE; VIRUS;

D O I：

10.1016/j.vascn.2010.09.006

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

Introduction: Various animal models are routinely used to evaluate the efficacy and toxicity of small interfering RNA (siRNA) therapeutics. Given that the most common measure of efficacy with siRNA therapeutics is mRNA knockdown, the development of a single assay for quantification of siRNA-mediated mRNA knockdown in multiple species would provide significant time and cost-savings during preclinical development. Methods and results: We have developed an assay targeting short consensus sequences of a particular mRNA in multiple species using the principles of a recently-reported stem-loop RT-qPCR method (Chen et al., 2005). The multi-species RT-qPCR assay is highly sensitive, reproducible, has a dynamic range of seven orders of magnitude, and it can be used to quantify a specific mRNA in crude tissue homogenates without the need for RNA purification. Compared to the limitations of conventional RT-qPCR assays, this assay provides a simple and robust tool for mRNA quantification to evaluate siRNA-mediated mRNA knockdown. Discussion: This assay can potentially become a routine method for mRNA quantification to evaluate siRNA-mediated mRNA knockdown. (C) 2010 Elsevier Inc. All rights reserved.

引用

页码：174 / 179

页数：6

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