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PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites
- Source :
- Frontiers in Genetics, Vol 9 (2018), Frontiers in Genetics
- Publication Year :
- 2018
- Publisher :
- Frontiers Media SA, 2018.
-
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.
- Subjects :
- 0301 basic medicine
Untranslated region
lcsh:QH426-470
3′ UTR
SNP
MiRNA binding
Computational biology
HeLa
03 medical and health sciences
0302 clinical medicine
microRNA
Methods
Genetics
Bioassay
Luciferase
Genetics (clinical)
miRNA
functional testing
high-throughput screening assays
biology
Three prime untranslated region
genetic variants
HEK 293 cells
biology.organism_classification
lcsh:Genetics
030104 developmental biology
Molecular Medicine
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 16648021
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- Frontiers in Genetics
- Accession number :
- edsair.doi.dedup.....cbea78cceb615a29c866b1f6e9800277
- Full Text :
- https://doi.org/10.3389/fgene.2018.00219