PASSPORT-seq: A novel high-throughput bioassay to functionally test polymorphisms in micro-RNA target sites

Joseph Ipe, Kimberly S. Collins, Yangyang Hao, Hongyu Gao, Puja Bhatia, Andrea Gaedigk, Yunlong Liu, Todd C. Skaar

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Next-generation sequencing (NGS) studies have identified large numbers of genetic variants that are predicted to alter miRNA-mRNA interactions. We developed a novel high-throughput bioassay, 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, we 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.

Original languageEnglish (US)
Article number219
JournalFrontiers in Genetics
Volume9
Issue numberJUN
DOIs
StatePublished - Jun 15 2018

Keywords

  • 3' UTR
  • Functional testing
  • Genetic variants
  • High-throughput screening assays
  • MiRNA
  • SNP

ASJC Scopus subject areas

  • Molecular Medicine
  • Genetics
  • Genetics(clinical)

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