Pmir report luciferase expression reporter vector

3′UTR target regions were amplified from 293T genomic DNA (human embryonic kidney cells) by polymerase chain reaction (PCR). Primer sequences were designed to incorporate SpeI and HindIII restriction enzyme sites (Supplementary Table S2). PCR products were subcloned into pMIR-REPORT luciferase expression reporter vector (Ambion, Grand Island, NY) to generate pMIR-3′UTR reporter. Point mutations were generated by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Columbia, MD, USA) according to the manufacturer's instructions (Supplementary Table S2). LNCaP or 293T cells were transfected with 20 nM of control or miRNA mimetic, 80 ng of pMIR-3′UTR reporter vector and 20 ng of reference pRL-CMV Renilla reporter vector. After 48 h, the reporter activity was assessed using a dual luciferase enzyme assay as previously described (12 (link)). Firefly luciferase signal was normalized against the internal control Renilla luciferase.