Luciferase Assay for miR-96 Binding

The predicted binding site for miR-96 on the 3′UTR of LCP1 was PCR-amplified as previously described [35 (link)] and cloned into the psiCHECK-2 plasmid (Promega). Negative control of LCP1 was achieved by substituting 3 nucleotides in the seed binding region of the cloned 3′UTRs using the QuikChange Lightning SDM kit (Agilent), as previously described [36 (link)]. HEK-293T and HeLa cells were seeded in 24-well plates supplemented with 10% FBS (GIBCO). Cells were transfected using Lipofectamine 2000 (Rhenium), 5 ng of the psiCHECK-2 relevant clone, 10 ng of pEGFP, and 485 ng miRVec containing the desired pre-miRNA. Twenty-four hours following transfection, lysates were extracted and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The Renilla luciferase results were normalized to the values of the firefly luciferase.