Experimental Validation of MicroRNA Targets Using a Luciferase Reporter System

Abstract

MicroRNAs (miRNAs) are a class of small noncoding transcripts that repress gene expression by pairing with their target messenger RNAs (mRNAs). The human genome codes for hundreds of different miRNAs and it is predicted that they target thousands of mRNAs involved in a wide variety of physiological processes such as development and cell identity. In animals, the identification of mRNA targets is complex because most miRNAs and their target mRNAs do not have exact or nearly exact complementarity. This tendency of animal miRNAs to bind their mRNA targets with imperfect sequence homology represents a considerable challenge to identifying miRNA targets. Computational algorithms based on conservation and experimental approaches based on expression profiles are flooding the literature with lists of candidate genes containing a large number of false-positive and false-negative predictions and indirect targets that cover the real list of direct targets for each miRNA. Currently, the only available tools to validate a sequence as a direct target of an miRNA are the systems based on a reporter gene carrying the candidate sequence. Here, an miRNA target validation reporter gene system based on the luminescence generated by the luciferase protein is described in detail, including the design of the reporter constructs, its expression in a model cell line and its measurement using a luciferase assay.