Measuring miRNA-mRNA Interactions via Luciferase Assay

We constructed sensor vectors by joining the region with or without a possible binding site from the 3′-UTR of human E2F3 (No. 4271-4650) with a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence of MIR34a. To generate sensor vectors with four mutations in the binding site of the 3′-UTR of human E2F3 (No. 4440-4462) for MIR34a, we mutated seed regions from CACTGCCA to CAAGTGCA (mt-E2F3, PrimeSTAR Mutagenesis Basal Kit; TaKaRa). The sensor vector with these mutations was submitted to Life Science Research Center, Gifu University, for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × 10⁵/well the day before transfection. The sensor vector (concentration, 0.5 μg/well) and 40 nM MIR34a or nonspecific control miRNA (Dharmacon) was used for the co-transfection of the cells with Lipofectamine RNAiMAX (Invitrogen). The measurement of the activities was described previously^{12 (link)}.