Reporter assays are used to test how endogenous miRNA/miRNP function is affected by exogenous miRNA transfections. H4 cells were first transfected with various miRNA precursors (negative control, miR-107, miR-124, miR-128, and miR-320) for 48 h A. Reporter plasmid constructs pRL-TK-Rluc carrying miR-320 MRE (Rluc-320), or its mutated sequence (Rluc-320mut) were co-transfected with calibrating plasmid pGL3 for an additional 24 h B. Luciferase activities were determined as previously described (Wang et al. 2008a,b). Luciferase activities, normalized to PGL-3 values, from each transfection were compared to that of negative control miRNA which represents endogenous miR-320 activity. Represented are three independent experiments each with three replicates. Student's t-tests were performed on the data set. “*,” P < 0.05; “**,” P < 0.01; “***,” P < 0.001.
