High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.
High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.
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DOI：
10.1093/nar/gkn725
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发表时间：
2008-12
影响因子：
14.9
通讯作者：
Vandesompele, Jo
中科院分区：
文献类型：
--
作者：
Mestdagh, Pieter;Feys, Tom;Bernard, Nathalie;Guenther, Simone;Chen, Caifu;Speleman, Frank;Vandesompele, Jo
微小RNA（microRNAs，miRNAs）是一类新出现的小分子非编码RNA，参与多种细胞过程。这一领域的研究正在加速，越来越多的miRNAs强调了对高通量和灵敏检测方法的需求。在这里，我们提出了成功的评估Megaplex逆转录格式的茎环引物为基础的实时定量聚合酶链反应（RT-qPCR）的方法来定量miRNA的表达。Megaplex反应可同时逆转录450种成熟miRNA，确保高通量检测。此外，引入互补DNA预扩增步骤显著降低了所需的输入RNA的量，甚至降低到单细胞水平。为了评估可能的预扩增偏倚，我们用Megaplex RT比较了三种不同癌细胞系中384种miRNA的表达，有或没有额外的预扩增步骤。所有三个样品对的归一化Cq值显示与细胞系之间差异miRNA表达的维持良好相关。此外，使用10 ng输入RNA的预扩增使得能够检测当单独使用Megaplex与400 ng输入RNA时不可检测的miRNA。RT-qPCR的高特异性以及上级灵敏度使该方法成为高通量miRNA表达谱分析的首选方法。
MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.
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影响因子：
64.8
作者：
Lu, J;Getz, G;Golub, TR
通讯作者：
Golub, TR
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
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作者：
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46.9
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通讯作者：
Wiemann, Stefan