The activity of miRNAs on target sequences have been studied using reporter assays where the target sites of interest are cloned into the 3′ UTR of a reporter gene followed by quantification of the reporter activity. Typical reporter assays also overexpress the miRNAs that are predicted to regulate the target site (Cloonan et al., 2008; Loya et al., 2009; Baccarini and Brown, 2010). Such overexpression, however, may not provide a physiological context to the miRNA–mRNA interaction. For example, high miRNA expression levels may force interactions with mRNAs that do not normally occur. They can also compete with the miRNA processing machinery or binding sites and alter normal miRNA function. In contrast, our assay was performed in the endogenous miRNA expression background, which provides a more physiologically relevant context of the results. In addition, we used multiple different cell lines to allow parallel testing to identify cell line-specific effects of the mirSNPs.
