Assaying dicer-mediated miRNA maturation by means of fluorescent substrates

Abstract

Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA maturation in a high-throughput format. The classical assay for miRNA maturation relies on radioactive labeling of a pre-miRNA and subsequent gel electrophoresis and autoradiography. Here we present a fluorescently labeled and quenched pre-miRNA beacon that can be ligated easily out of two single labeled RNA strands. Upon Dicer cleavage of the beacon, fluorophore and quencher are separated, which results in an increase of fluorescence over time. Unlike 32 P-labeled probes, our fluorescently labeled pre-miRNA beacon is stable for at least 5 years under storage conditions. Dicer or miRNA maturation assays can be easily performed in a 384-well plate format, consuming less than 1 pmol of RNA beacon per reaction. © 2014 Springer Science+Business Media, New York.