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Quantitation of microRNAs using a modified Invader assay

被引：137
作者：
Allawi, HT
Dahlberg, JE
Olson, S
Lund, E
Olson, M
Ma, WP
Takova, T
Neri, BP
Lyamichev, VI
机构：
[1] Third Wave Technol Inc, Madison, WI 53719 USA
[2] Univ Wisconsin, Sch Med, Dept Biomol Chem, Madison, WI 53706 USA
来源：
RNA | 2004年 / 10卷 / 07期
关键词：
miRNA; siRNA; RNAi; structure-specific 5'-nuclease; 2'-O-methyl; Invader assay; cell lysates;
D O I：
10.1261/rna.5250604
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.
引用
收藏
页码：1153 / 1161
页数：9
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