Dual Luciferase Assay for IRES Activity

After BHK-21 cells were knocked down or overexpressed, the cells were transfected with the corresponding bicistronic reporter plasmid, psiCHECK or psiCHECK-FMDV, respectively. At 24 h post-transfection, the cells were lysed by the special cell lysate buffer for dual luciferase reporter assay, and the signal intensities of Firefly luciferase (Fluc) and Renilla luciferase (RLuc) were detected with the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions. Monocistronic luciferase activity was measured in the rabbit reticulocyte in vitro translation system [rabbit reticulocyte lysate (RRL), Promega] as previously described (29 (link)). The template with pGL3-FMDV IRES-FLuc gene for in vitro translation was synthesized by Azenta Life Sciences (Suzhou, China). RALY (50 pmol) or a non-specific control protein, BSA, and 0.5 µg of template RNA in vitro transcript were added to 200 µL of the reaction system and incubated at 30°C for 40 min, and luciferase activity was immediately determined with Steady-Glo (Promega).