Dual luciferase gene reporter assays to study miRNA Function

Abstract

This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression.