Next-generation sequencing (NGS) studies have identified large numbers of genetic variants that are predicted to alter miRNA–mRNA interactions. Researchers from Indiana University School of Medicine recently developed a novel high-throughput bioassay using OligoMix, PASSPORT-seq, that can functionally test in parallel 100s of these variants in miRNA binding sites (mirSNPs). The results are highly reproducible across both technical and biological replicates.

The utility of the bioassay was demonstrated by testing 100 mirSNPs in HEK293, HepG2, and HeLa cells. The results of several of the variants were validated in all three cell lines using traditional individual luciferase assays. Fifty-five mirSNPs were functional in at least one of three cell lines (FDR ≤ 0.05); 11, 36, and 27 of them were functional in HEK293, HepG2, and HeLa cells, respectively. Only four of the variants were functional in all three cell lines, which demonstrates the cell-type specific effects of mirSNPs and the importance of testing the mirSNPs in multiple cell lines. Using PASSPORT-seq, researchers functionally tested 111 variants in the 3′ UTR of 17 pharmacogenes that are predicted to alter miRNA regulation. Thirty-three of the variants tested were functional in at least one cell line.



Cell line-specificity of functional mirSNPs in pharmacogenes. Venn diagram showing the number of unique and overlapping functional mirSNPs in the four cell lines tested.

J. Ipe, K. S. Collins, Y. Hao, H. Gao, P. Bhatia, A. Gaedigk, Y. Liu, T. C. Skaar (2018) PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites Front. Genet. doi: 10.3389/fgene.2018.00219 [article]

microRNA Functions in the Regulation of Wood Formation in Trees MPE-seq - Multiplexed Primer Extension sequencing with microarray synthesized oligos