Dual luciferase gene reporter assays to study miRNA function.
Fiche publication
Date publication
janvier 2015
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr REDERSTORFF Mathieu
Tous les auteurs :
Clément T, Salone V, Rederstorff M
Lien Pubmed
Résumé
This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression.
Mots clés
Cloning, Molecular, Gene Silencing, physiology, Genes, Reporter, genetics, HeLa Cells, Humans, Luciferases, genetics, Luminescent Measurements, methods, MicroRNAs, metabolism, RNA, Messenger, genetics, Untranslated Regions, genetics
Référence
Methods Mol. Biol.. 2015 ;1296:187-98