title: Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay.
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.
authors.affiliation: School of Life Sciences, Arizona State University; Biodesign Institute, Arizona State University.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Wolter
authors.affiliation: School of Life Sciences, Arizona State University; Biodesign Institute, Arizona State University.
authors.forename: Kasuen
authors.initials: K
authors.lastname: Kotagama
authors.affiliation: Biodesign Institute, Arizona State University.
authors.forename: Cody S
authors.initials: CS
authors.lastname: Babb
authors.affiliation: School of Life Sciences, Arizona State University; Biodesign Institute, Arizona State University; mangone@asu.edu.
authors.forename: Marco
authors.initials: M
authors.lastname: Mangone
mesh_terms: D020413:3' Untranslated Regions; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D012333:RNA, Messenger; D008156:Luciferases