Dual luciferase gene reporter assays to study miRNA function.

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

https://www.ncbi.nlm.nih.gov/pubmed/25791601

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