Detection of miRNA targets in high-throughput using the 3'LIFE assay

Justin M. Wolter, Kasuen Kotagama, Cody S. Babb, Marco Mangone

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Luminescent Identification of Functional Elements in 3’UTRs (3’LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3’UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3’LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3’LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3’UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Original languageEnglish (US)
Article numbere52647
JournalJournal of Visualized Experiments
Volume2015
Issue number99
DOIs
StatePublished - May 25 2015

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MicroRNAs
Assays
Throughput
Luciferases
Cell culture
DNA
Genomic Library
Genes
Robots
Transfection
Buffers
Liquids
Cell Culture Techniques
Costs
Costs and Cost Analysis
Messenger RNA

Keywords

  • 3' untranslated region
  • Cancer
  • High-throughput
  • Issue 99
  • Luciferase assay
  • MicroRNA
  • Molecular biology
  • Post-transcriptional gene regulation
  • Transfection

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Detection of miRNA targets in high-throughput using the 3'LIFE assay. / Wolter, Justin M.; Kotagama, Kasuen; Babb, Cody S.; Mangone, Marco.

In: Journal of Visualized Experiments, Vol. 2015, No. 99, e52647, 25.05.2015.

Research output: Contribution to journalArticle

Wolter, Justin M. ; Kotagama, Kasuen ; Babb, Cody S. ; Mangone, Marco. / Detection of miRNA targets in high-throughput using the 3'LIFE assay. In: Journal of Visualized Experiments. 2015 ; Vol. 2015, No. 99.
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