Fluoroskan ascent fl instrument

To determine if miR-218-5p targets the ELOVL5 3′UTR, DF1 cells were seeded in 12-well plates in triplicate and transfected with a wild-type or mutant construct in serum-free DMEM medium. After 6 h of transfection, the medium was changed. Cells were harvested and lysed 48 h after transfection. Luciferase activity was measured using the Dual-Luciferase^® Report Assay System (Promega, Maddison, WI, USA) on a Fluoroskan Ascent FL instrument (Thermo Fisher Scientifc, Shanghai, China). Renilla luciferase activity was normalized to Firefly luciferase activity. And this transfection experiment was performed in triplicate wells and repeated at least three times.

To determine the interaction of miR-34a-5p and its potential target gene ACSL1, DF1 cells were seeded in 24-well plates and co-transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in triplicate with 500 ng of the wild-type or mutant-type plasmid and a final concentration of 80 nM miR-34a-5p mimics or miR-34a-5p mimics NC in serum-free medium. At 48 h after transfection, the cells were washed with 1× phosphate-buffered saline (PBS) (Solarbio, Beijing, China) three times and lysed with passive lysis buffer (PLB). Then, the Renilla luciferase and firefly luciferase activities were measured using a Dual-Luciferase^® Reporter Assay System (Promega, Maddison, WI, USA) on a Fluoroskan Ascent FL instrument (Thermo Fisher Scientifc, Shanghai, China). Renilla luciferase activity served as an internal control to normalize firefly luciferase activity.