Dual luciferase assay kit

Luciferase reporter gene recombinant plasmids were inserted with the sequences of wild-type (WT) and mutated type (MUT) circSCNA, WT and MUTSCNA 3’-untranslated region (3’-UTR). MiR-7 mimics or control were co-transfected with WT and MUT circSCNA into the 293 cell line (BeNa Culture Collection, Beijing, China) using Lipofectamine 2000 (Invitrogen). Luciferase Dual Assay Kit (Thermo Fisher Scientific) was used for dual-luciferase reporter gene assay 48 h after cells were transfected.

Transcriptional activity of the HSPA5 promoter was measured using a luciferase dual assay system. The HSPA5 promoter region containing the TEAD consensus binding motif was amplified by PCR using the designed primers, forward 5′-GGACTAGTCCACGGTAGGCTTTCAG-3′ and reverse 5′-CGCGGATCCCTTGCCAGCCAGTTG-3′, and cloned into the pMCS-Cypridina luciferase reporter vector (Thermo Fisher, Pittsburgh, PA, USA). The pTK-Red Firefly luciferase vector (Thermo Fisher, Pittsburgh, PA, USA) was used as an internal control. Polyjet transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) was used for co-transfection of the two plasmids. Luciferase activity was detected using a luciferase dual assay kit (Thermo Fisher, Pittsburgh, PA, USA) according to the manufacturer’s instructions. Firefly luciferase activity (encoded by the control plasmid) was used to normalize Cypridina luciferase activity (encoded by the experimental plasmid).