Validation of microRNA Target Genes Using Luciferase Reporter assay and Western Blot Analysis.

MicroRNA s regulate gene expression by binding to the 3'untranslated region (UTR) of the mRNA of their target genes. Identification of microRNA target genes enables the determination of their functional role in the cells. A single microRNA can target multiple genes, all of which have a microRNA binding site in their 3' UTR. Putative target genes can be identified using target prediction software and gene expression analysis of microRNA expressing cells. The validation of the putative target genes is carried out using the luciferase reporter assay and western blot analysis. This chapter describes the protocol for using these techniques for validation of putative microRNA target genes.