Pmirglo dual luciferase mirna target expression vector pmirglo vector

For construction of the AHR 3′-UTR-Luc reporter plasmid, a fragment of the 3′-untranslated region (3′-UTR) of the AHR gene was synthesized and cloned into pUC57 (Bio-Basic Inc., Amherst, NY, USA) within the SacI and SalI restriction sites. The pmirGLO Dual-Luciferase miRNA target expression vector (pmirGLO vector) containing both the firefly luciferase (Fluc) gene and the Renilla luciferase (Rluc) gene was purchased from Promega (Madison, WI, USA). The AHR 3′-UTR was then subcloned by 1st BASE Services (First BASE Laboratories, Selangor, Malaysia), directly downstream from a Fluc gene under the control of the PGK promoter, and Rluc gene under the control of an SV40 promoter (as a transfection control) within the SacI and SalI restriction sites. MDA-MB-231 and MCF-7 cells were cotransfected with 50 ng reporter plasmids and 25 nM miR-548m mimics and its control for 48 h. The luciferase activity was determined with Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instruction. Luciferase activity was measured with SpectraMax L luminometer (Molecular Devices). Normalized data were calculated as the ratio of Rluc/Fluc activities.