PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites
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)....
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-06-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fgene.2018.00219/full |
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| author | Joseph Ipe Kimberly S. Collins Kimberly S. Collins Yangyang Hao Yangyang Hao Hongyu Gao Hongyu Gao Puja Bhatia Andrea Gaedigk Yunlong Liu Yunlong Liu Todd C. Skaar |
| author_facet | Joseph Ipe Kimberly S. Collins Kimberly S. Collins Yangyang Hao Yangyang Hao Hongyu Gao Hongyu Gao Puja Bhatia Andrea Gaedigk Yunlong Liu Yunlong Liu Todd C. Skaar |
| author_sort | Joseph Ipe |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T22:53:08Z |
| format | Article |
| id | doaj.art-03a6dbe8a747469f9abd77c00d971d9d |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-21T22:53:08Z |
| publishDate | 2018-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-03a6dbe8a747469f9abd77c00d971d9d2022-12-21T18:47:30ZengFrontiers Media S.A.Frontiers in Genetics1664-80212018-06-01910.3389/fgene.2018.00219335941PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target SitesJoseph Ipe0Kimberly S. Collins1Kimberly S. Collins2Yangyang Hao3Yangyang Hao4Hongyu Gao5Hongyu Gao6Puja Bhatia7Andrea Gaedigk8Yunlong Liu9Yunlong Liu10Todd C. Skaar11Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United StatesDivision of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United StatesDepartment of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, United StatesDepartment of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United StatesCenter for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, United StatesDepartment of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United StatesCenter for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, United StatesDivision of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United StatesDivision of Clinical Pharmacology, Toxicology and Therapeutic Innovation, Children’s Mercy Kansas City, Kansas City, MO, United StatesDepartment of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United StatesCenter for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, United StatesDivision of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United StatesNext-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.https://www.frontiersin.org/article/10.3389/fgene.2018.00219/fullSNPfunctional testinggenetic variantsmiRNAhigh-throughput screening assays3′ UTR |
| spellingShingle | Joseph Ipe Kimberly S. Collins Kimberly S. Collins Yangyang Hao Yangyang Hao Hongyu Gao Hongyu Gao Puja Bhatia Andrea Gaedigk Yunlong Liu Yunlong Liu Todd C. Skaar PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites Frontiers in Genetics SNP functional testing genetic variants miRNA high-throughput screening assays 3′ UTR |
| title | PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites |
| title_full | PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites |
| title_fullStr | PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites |
| title_full_unstemmed | PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites |
| title_short | PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites |
| title_sort | passport seq a novel high throughput bioassay to functionally test polymorphisms in micro rna target sites |
| topic | SNP functional testing genetic variants miRNA high-throughput screening assays 3′ UTR |
| url | https://www.frontiersin.org/article/10.3389/fgene.2018.00219/full |
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