Prl tk control vector expressing renilla luciferase

4 × 10⁴ 293 cells were co-transfected in 24-well dishes with the pGL3-KRAS 3’-UTR luciferase reporter construct or with the mutated KRAS 3’-UTR luciferase vector, together with the Renilla luciferase plasmid and with the RNA oligonucleotides (Ambion), using lipofectamine plus (Ambion). The pRL-TK control vector expressing Renilla luciferase (Promega) was used to normalize cell number and transfection efficiency. Luciferase activity was measured as previously reported [25 (link)].

For dual-luciferase reporter assay, 3 × 10⁵ NIH3T3 cells were co-transfected in 6-well plates with the pGL3- Igf2 or the pGL3-H19 luciferase reporter vectors, together with the Renilla luciferase plasmid and miRNA precursors or a control no-targeting scrambled oligonucleotides (Thermo Fisher Scientific Inc), using siPORT neoFX Transfection Agent (Thermo Fisher Scientific Inc). The pRL-TK control vector expressing Renilla luciferase (Promega) was used for normalization of cell number and transfection efficiency. Luciferase activity was measured 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega) with a Lumat LB 9507 apparatus (Berthold Technologies).

The TargetScan database (http://www.targetscan.org/) was retrieved to search for potential targets of miR-541-3p, which indicated HSP27 to be a target of miR-541-3p. To further investigate whether miR-541-3p directly targeted HSP27, we utilized dual-luciferase reporter assay. The 3′-UTR of HSP27 was amplified using PCR, and the 3′-UTR mutation product of HSP27 (mut-3′-UTR) was synthesized by site-directed mutation. After cleavage with restriction enzymes, the amplified PCR product was cloned into the polyclonal site of the reconstructed pGL3 expression vector. The pRL-TK control vector expressing Renilla luciferase (Promega, Madison, WI, USA) was utilized for normalization of transfection efficiency. Finally, the luciferase activity was detected using Dual-Luciferase® Reporter assay kits (Promega, Madison, WI, USA).