Experimental miRNA target validation.

In the recent past, microRNAs (miRNAs) have gained significant attention as potent regulators of gene expression. These small noncoding RNA molecules are currently of major interest when investigating regulatory circuits of the cell. After identification of potential miRNA-target gene interactions (e.g., using computational algorithms), biomolecular validation is necessary. In the current chapter, we present a protocol for validation of an miRNA target interaction implying cloning of a dual-luciferase miRNA target expression vector, transfection of cells with this vector and a precursor miRNA (pre-miRNA), and the subsequent luciferase assay.