Real-time quantification of miRNAs and mRNAs employing universal reverse transcription

Introduction MicroRNAs have been identified as a new level of eukaryotic gene regulation. These endogenous ∼22 nucleotide (nt) sequences are variably expressed in a manner that is specific to cell types, developmental stages and diseases (such as cancer). Because of their small size and variable expression levels, detection and quantification is technically challenging; various techniques have been developed to address these issues. We have modified a technique originally designed to specifically amplify mRNA and exclude pseudogene co-amplification to the real-time quantification of miRNAs. The modified technique involves the enzymatic addition of a polyA tail and the sequential hybridization of a universal RT-primer followed by reverse transcription. Transcript-specific forward primers can then be used to amplify a number of miRNAs as well as mRNA, from the same sample. We have used this technique to quantify relative miRNA expression using GAPDH mRNA as an internal standard. Relative quantification against a ubiquitously expressed miRNA, or multiplexing, is also possible, as all miRNAs in a sample are reverse transcribed. In addition, the relative quantification of both a miRNA and its predicted mRNA target can be assessed in the same sample. This allows identification of miRNA/mRNA interaction that results in cleavage or degradation of the target. The method is straightforward, needs no RNA size fractionation and involves routine enzymatic manipulations.