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Nucleic Acids Research Advance Access published online on October 21, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn725
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA

Pieter Mestdagh1, Tom Feys1, Nathalie Bernard2, Simone Guenther2, Caifu Chen2, Frank Speleman1 and Jo Vandesompele1,*

1Center for Medical Genetics, Ghent University Hospital, 9000 Ghent, Belgium and 2Applied Biosystems, Foster City, 94404 CA, USA

*To whom correspondence should be addressed. Tel: +32 9 332 5187; Fax: +32 9 332 6549; Email: joke.vandesompele{at}ugent.be

Received July 22, 2008. Revised September 3, 2008. Accepted October 1, 2008.

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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