Validating microRNA Target Transcripts Using Zebrafish Assays

Hundreds of tiny noncoding RNAs known as microRNAs (miRNAs) have been identified in the genomes of plants and animals. Studies are increasingly demonstrating that individual miRNAs are important in normal development and physiology. miRNAs are regulators of gene expression that bind target mRNAs and modulate their translation and turnover. The specificity of this regulation is achieved by partial sequence complementarity between the miRNA and its target mRNA. Understanding which mRNAs are targeted by each particular microRNA is critical to an understanding of the biologic role of any particular miRNA. Bioinformatic approaches can be used to predict mRNAs that may be miRNA targets, but each of these predictions requires experimental validation. We describe a method for a reporter assay based on a fluorescence intensity readout that uses transient techniques in zebrafish to easily deliver the reporter assay components. In addition, we describe a rigorously controlled strategy for determining the bona fide miRNA binding sites in the 3′UTR of mRNAs.