Pcdna3.1 containing a hygromycin resistance gene

HeLa cells were transfected with the psiCHECK-2 vector (Promega) and the pcDNA3.1 containing a hygromycin resistance gene (Thermo Fisher Scientific). HeLa Cells were grown in the presence of 0.5 mg mL⁻¹ hygromycin for 1 week. Stable HeLa-psiCHECK-2 cells expressing both firefly and Renilla luciferases were grown in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 0.25 mg mL⁻¹ hygromycin and 10% bovine serum (BS) at 37 °C. 24 hours prior to transfection of siRNAs, HeLa-psiCHECK-2 cells (8.0 × 10⁴ mL⁻¹) were grown in a 96-well plate (100 μL per well). The cells were transfected with siRNAs targeting the Renilla luciferase gene using lipofectamine RNAiMAX in opti-MEM reduced serum medium. Transfection without siRNAs was used as a control. The cells were incubated for 1 hour, D-MEM (50 μL) containing 10% BS was added to each well and cells were further incubated for another 24 hours. The activities of firefly and Renilla luciferases in the cells were measured using the Dual-Luciferase Reporter Assay System (Promega) according to a manufacture's protocol. The activity of Renilla luciferase was normalized by the firefly luciferase activity. The results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean ± SD.