A Novel Method to Detect Functional MicroRNA Targets

MicroRNA (miRNA) molecules are non-coding RNAs, 19 to 24 nt in length that have been identified recently as important regulators of gene expression. Several computational methods have been developed to describe the target recognition mechanism by miRNA. We propose here a novel method to detect miRNA–mRNA complexes in eukaryotic cells. As a first step, we synthesize cDNA on an mRNA template using miRNAs as the endogenous cytoplasmic primer. This step extends miRNA and overcomes the problem of low complementary binding of miRNAs to their targets. Purified hybrid 3′-cDNA–miRNA-5′ molecules are used in a second round of reverse transcription to anneal to target mRNA in a highly gene-specific manner. The 5′-end analysis of these cDNA molecules demonstrated that primers for cDNAs were “signatures” of miRNA molecules, and over-expression of their full-length mature miRNAs resulted in functional inhibition of target protein expression.