Joseph Ipe, Kimberly S. Collins, Yangyang Hao, Hongyu Gao, Puja Bhatia et al.
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.
Joseph Ipe, Kimberly S. Collins, Yangyang Hao, Hongyu Gao, Puja Bhatia, Andrea Gaedigk, Yunlong Liu, Todd C. Skaar. PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites. 2018-06-15. https://doi.org/10.3389/fgene.2018.00219
The pharmacogene library contained reference and alternate 63-nt 3′-UTR inserts for 111 mirSNPs across 17 pharmacogenes. The pooled pIS-0 reporter was transiently assayed in HepG2, and variant effects are reported as percent change in reporter mRNA expression relative to the reference allele.
The pharmacogene library contained reference and alternate 63-nt 3′-UTR inserts for 111 mirSNPs across 17 pharmacogenes. The pooled pIS-0 reporter was transiently assayed in HepaRG, and variant effects are reported as percent change in reporter mRNA expression relative to the reference allele.
The pharmacogene library contained reference and alternate 63-nt 3′-UTR inserts for 111 mirSNPs across 17 pharmacogenes. The pooled pIS-0 reporter was transiently assayed in HEK293, and variant effects are reported as percent change in reporter mRNA expression relative to the reference allele.
The pharmacogene library contained reference and alternate 63-nt 3′-UTR inserts for 111 mirSNPs across 17 pharmacogenes. The pooled pIS-0 reporter was transiently assayed in HeLa, and variant effects are reported as percent change in reporter mRNA expression relative to the reference allele.
The 200-construct library contained reference and alternate 63-nt miRNA-binding-site inserts for 100 mirSNPs. The pooled pIS-0 3′-UTR reporter was transiently transfected into HEK293 and five biological replicates were quantified by barcode sequencing relative to four plasmid-input replicates.
The 200-construct library contained reference and alternate 63-nt miRNA-binding-site inserts for 100 mirSNPs. The pooled pIS-0 3′-UTR reporter was transiently transfected into HeLa and five biological replicates were quantified by barcode sequencing relative to four plasmid-input replicates.
The 200-construct library contained reference and alternate 63-nt miRNA-binding-site inserts for 100 mirSNPs. The pooled pIS-0 3′-UTR reporter was transiently transfected into HepG2 and five biological replicates were quantified by barcode sequencing relative to four plasmid-input replicates.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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