Evaluating Single-Nucleotide Variants in MicroRNA Targeting Sites and Mature MicroRNA In Vitro Cell Culture by Luciferase Reporter Gene Assays

Abstract

MicroRNAs (miRs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. Single-nucleotide variants (SNVs) in primary miRs (pri-miRs), precursor miRs (pre-miRs), promoters of pri-miRs, and seed regions can affect miR stability or processing, may influence mature miR expression, and can affect target gene identification, respectively. The present protocol tests the binding and activity of miRs on 3'-UTR target sequences based on the expression of luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miR of interest to test in vitro cell culture assay.

Keywords: 3′-UTR; In vitro cell culture; Luciferase assay; MicroRNA activity.