Hblumi dual luciferase reporter assay kit

By using psi-CHECK-2 vector, a total of four dual-luciferase reporters, including SNAI3-AS1 promoter, Nrf2 promoter, Nrf2 mRNA 3’UTR_WT, and Nrf2 mRNA 3’UTR_Mut, were synthesized by GeneCreate Biological Engineering. Detailed sequences were listed in Supplementary Table 4–6. In brief, cells seeded in 24-well plates with 60–80% confluency were transfected with dual-luciferase reporters. Forty-eight hours after transfection, renilla and firefly luciferase activities in each well were detected by using HBLumi Dual-luciferase reporter assay kit (Hanbio, Shanghai, China) according to the manufacturer’s instructions.
To evaluate the RNA stability, cells were treated by actinomycin D (5 μg/ml, M4881, AbMole BioScience) for 0, 2, 4, 6 h. The total RNA was extracted by Trizol Reagent and analyzed by RT-qPCR as described above. The results were normalized to the values measured at 0 h.

The wild-type or mutant SNHG10 or BIN1-3’-UTR containing the predicted binding sites of miR-200a-3p was subcloned into an HBLumi Dual-luciferase reporter assay kit (Hanbio, Shanghai, China). The luciferase reporter plasmids were co-transfected into EOC cells with miR-200a-3p mimic or the negative control. The relative luciferase activity was measured according to the manufacturer’s instructions. All transfection assays were carried out in triplicate.

Sequences for wild-type (WT) or mutant (Mut) NORAD or the 3′-UTR of MTDH containing the predicted binding sites for miR-224-3p were subcloned into a psiCHECK-2 vector (Hanbio Biotechnology, Shanghai, China). The luciferase reporter plasmids were co-transfected into ESCC cells with a miR-224-3p mimic or negative control (NC). After 48 h, the cells were collected and counted. A total of 5 × 10⁵ cells were used to measure luciferase activity. Relative luciferase activity was measured by using an HBLumi Dual-luciferase reporter assay kit (Hanbio Biotechnology) according to the manufacturer’s instructions. Renilla luciferase activity was normalized to firefly luciferase activity. To evaluate the effect of the miR-224-3p mimic on firefly luciferase activity, the miRNA mimic NC group was used as control. All assays were carried out in triplicate. The mutant sequences of NORAD and the MTDH-3′-UTR were created by using the FAST Site-Directed Mutagenesis Kit (Tiangen, Beijing, China).