Firefly luciferase assay kit

According to the online prediction of miR-128 binding sequence in the 3ʹUTR of Axin1 mRNA, the target (WT) sequence and mutant (mut) sequence were verified, synthesized, and constructed into a pGL3-reporter plasmid as described.^{24 (link)} The Axin1-luc plasmids were then packaged into rAAV9 expression system and co-transfected with miR-128 mimics or control plasmids into mice as described above. Four weeks after in vivo gene transfer, cardiomyocytes were isolated and cell lysates were prepared with a Firefly Luciferase Assay Kit (Beyotime, China) following the manufacturer’s instructions. Lysates were then analyzed for luciferase activity using a Glomax20/20 luminometer (Promega, WI). The relative luciferase value was normalized to the level of renilla luciferase activity in each sample.

RAW 264.7 cells (1 × 10⁵/mL) were cultured in a 24-well plate and transfected with plasmids pGL-luc2P/NF-κBRE (Promega, USA) and pAP1-luc and pIFN-β-luc (Genepharma, China) using X-tremeGENE HP DNA Transfection Reagent (Roche) for 24 h. Then, the plate was added with LPS for 6 h of incubation treatment. The luciferase activity was analyzed with the Firefly Luciferase Assay Kit (Beyotime) and detected in a multimode reader (Thermo). Relative luciferase light units were normalized to untreated cells.

The target site sequence of NLRP3 mRNA 3′-UTR wild type (WT) and the mutant sequence (MUT) were synthesized. The pmiR-RB-REPORTTM plasmid (RiboBio, Guangzhou, Guangdong, China) was digested using restriction endonuclease, and the synthesized target gene sequence WT and MUT were, respectively, inserted into pmiR-RB-REPORTTM vectors. The correctly sequenced luciferase reporter plasmid WT and MUT were transfected with mimic-NC or miR-204-5p mimic into 293T cells. After 48 h of transfection, cells were harvested, lysed, and centrifuged for 3–5 min. The relative luminescence unit (RLU1) of firefly luciferase in the supernatants was detected using Firefly Luciferase Assay kit (RG005, Beyotime, Shanghai, China), and relative fluorescent value was obtained with the relative luminescence unit (RLU2) of Ranilla luciferase as an internal control. The experiment was repeated three times. The binding relation between Kcnq1ot1 and miR-204-5p was verified in the same way. The wild-type sequence or mutant sequence of Kcnq1ot1 was, respectively, inserted into pGL3-reporter vectors and transfected with miR-204-5p mimic or mimic-NC into the HEK293T cells, followed by detecting luciferase activity.