Dual-Luciferase Assay for miRNA Regulation

pRL-Con contains the humanized Renilla Luciferase coding region. pRL-3xBulge-miR122 contains three miR-122 binding sites downstream of Renilla Luciferase (RL) coding region. pGL3FF (Promega) encodes a Firefly Luciferase (FL) gene under the SV40 promoter. The RL-Con/RL-3xBulge-miR122 was cotransfected with FL plasmids in 6-well plates, as described earlier [58 (link)]. The cells were then split after 24 h into plates with thin coatings of Collagen and Matrigel (with the lowest concentrations to avoid cell lysis problems) or into Control plates. The cells were then allowed to grow for 72 h before the relative activities were measured using a Dual-Luciferase Assay Kit (Promega, Madison, WI) following the supplier’s protocol on a Mithras Luminometer (Berthold Technologies). Two independent experiments with two technical replicates were assayed for each condition (with individual Controls).