MyD88 wild-type and its corresponding mutant were built into a pGL3-basic luciferase vector (2 μg/mL; Shanghai GenePharma, Shanghai, China) to generate the luciferase reporter plasmids. Cells at the third passage were inoculated onto 24 well plates at a density of 4 × 104 cells/well and cultured at 37°C. When the cells reached ~ 70% confluence, they were co-transfected with luciferase reporter plasmids, miR-143-3p mimic, or NC mimic using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer’s instructions. Luciferase activity was examined using a dual-luciferase reporter assay kit (cat. no. E1910; Promega Corporation, Madison, WI, USA) following transfection for 24 h [29 (link)].
Protocol detail
Luciferase Reporter Assay for miR-143-3p
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Corresponding Organization : Lanzhou University
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Variable analysis
independent variables
- MyD88 wild-type
- MyD88 mutant
- Luciferase activity
- Cells at the third passage
- Cell density (4 × 10^4 cells/well)
- Cell culture temperature (37°C)
- Cell confluence (~ 70%)
- Transfection reagent (Lipofectamine 2000)
- Transfection duration (24 h)
- miR-143-3p mimic
- NC mimic
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