Abstract
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.
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Acknowledgment
This work was supported by the Centre National pour la Recherche Scientifique, the Université de Lorraine, the Région Lorraine and the Ligue nationale pour la recherche contre le cancer (comités 54 et 57).
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Clément, T., Salone, V., Rederstorff, M. (2015). Dual Luciferase Gene Reporter Assays to Study miRNA Function. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 1296. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2547-6_17
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DOI: https://doi.org/10.1007/978-1-4939-2547-6_17
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2546-9
Online ISBN: 978-1-4939-2547-6
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