Multiplexing RT-PCR for High-Throughput miRNA Profiling

Abstract

While the RT-PCR techniques introduced in Chaps. 5–7 are in general considered low throughput methods, they can be modified and upgraded to medium throughput miRNA detection methods. In 2006, Lao and colleagues from the Applied Biosystems, Foster City (CA, USA) investigated the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple miRNAs in small sample sizes down to the amount of RNA found in a single cell (Biochem Biophys Res Commun 343:85–89, 2006). In the same year, Surani and colleagues from Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge (Cambridge, UK) (Nucleic Acids Res 34:e9, 2006; Nat Protoc 1:1154–1159, 2006) developed a Single Cell Stem-Looped Real-Time PCR (SC-SL-RT-PCR) protocol for the detection of the expression profile of 220 mature miRNAs in a single embryonic stem cell, cell by stem-looped real-time PCR. More recently, Mestdagh et al. from the Center for Medical Genetics, Ghent University Hospital (Ghent, Belgium) (Nucleic Acids Res 36:e143, 2008) presented the successful evaluation of the Megaplex (450 mature miRNAs) reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. In the meantime, Schmittgen et al. (Mol Biol 429:89–98, 2008) from College of Pharmacy, Ohio State University (Columbus, OH, USA) also reported an example of profiling the expression of over 200 miRNA precursors (pre-miRNAs) using high-throughput real-time PCR. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling (Wark et al. 2008). While PCR will never rival the throughput of microchip arrays, in situations where one is interested in assaying several hundreds of genes, high throughput, real-time PCR is an excellent alternative to microchip arrays. Moreover, multiplexing RT-PCR is quantitative for miRNA expression detection.