Dual luciferase assay kit

Mutant versions of the miR-146a-5p binding site sequences in JAZF1 were synthesized and inserted into the psiCHECK2 luciferase reporter vector (Hanbio Biotechnology) to create JAZF1 wild-type (JAZF1-WT) and mutant (JAZF1-Mut). The dual-luciferase assay was conducted using a dual-luciferase assay kit (Hanbio Biotechnology). The relative luciferase activity of fireflies and kidney worms was measured using SpectraMax M5e.

The sequences of circ3823, TCF7–3’UTR and their miR-30c-5p binding sites mutant versions were synthesized and add to luciferase reporter vector psiCHECK2 (Hanbio Biotechnology, Wuhan, China), named circ3823-WT, circ3823-Mut, TCF7-WT and TCF7-Mut, respectively. The experimental steps follow the manufacturer’s protocols of Dual Luciferase Assay Kit (Hanbio Biotechnology, Wuhan, China). The relative luciferase activity was examined by Full-wavelength multifunctional enzyme label tester SpectraMax M5e (Molecular Devices, Shanghai, China).

The anticipated miR-502-5p attachment locations within the complete wild-type (WT) circSLCO1B3 sequence, the untranslated region of HOXC8, and the specified mutant circSLCO1B3 and HOXC8 sequences (Mut) were created and inserted into the psi-CHECK-2 dual-luciferase reporter vector (Hanbio Biotechnology, China) separately. The trial is conducted following the guidelines provided by the manufacturer for the Dual Luciferase Assay Kit (Hanbio Biotechnology, China). 293 T cells were transfected with plasmids following a 48-hour interval, the cells were lysed and gathered. The firefly luciferase activities were assessed by the SpectraMax M5e, a versatile enzyme label tester that measures full-wavelength, from Molecular Devices in Shanghai, China.