The traditional luciferase reporter assay is useful in low-throughput experiments, but is not a practical and cost-effective method to test the 1000s of mirSNPs identified at a genome-wide scale. As a novel approach, we modified the luciferase reporter assay to develop PASSPORT-seq that can functionally test 100s of mirSNPs in parallel. Since one of the mechanisms of miRNA regulation is by degrading mRNA, this assay was specifically designed to evaluate the impact of genetic variation in miRNA binding sites on mRNA expression. We recognize that miRNAs also alter mRNA translation, however measuring protein levels does not distinguish between the impact on mRNA vs. translation and thus would not provide the same mechanistic insights. We identified 100 variants in predicted seed sequences of miRNA binding sites, and cloned the binding sites into the pIS-0 luciferase plasmid; each contained either the reference or variant nucleotide of 100 selected mirSNPs. The pool of the resulting 200 plasmids was then transfected into three cell lines and the luciferase gene expression measured by NGS. A difference in mRNA expression between the reference and the variant plasmids indicated a functional mirSNP.
