The binding sites between miR-26a and SLC7A11 mRNA were predicted by the miRDB and targetScan online website. LX2 cells were seeded into the 24‑well plates and grown with 5% CO2 at 37°C. When the cells’ confluency reached 70–80%, the transfection was conducted. Wild-type SLC7A11 (SLC7A11-WT) and mutant SLC7A11 (SLC7A11-MUT) were sub-cloned into pGL3-Basic luciferase vector (2 μg/mL, GenePharma) and then transfected into LX2 cells with miR-26a-mimic or NC-mimic using Lipofectamine™ 3000 (Cat#: L3000001, Invitrogen). Forty-eight hours later, luciferase activity was measured by a Dual Luciferase Reporter Gene Assay Kit (Cat#: RG027, Beyotime) based on the instruction for use.
Pgl3 basic luciferase vector
Manufactured by GenePharma
Sourced in China
The PGL3-Basic luciferase vector is a plasmid designed for use in reporter gene assays. It contains the firefly luciferase gene as the reporter, which can be used to measure gene expression levels in cells. The vector provides a basic backbone for cloning and expressing target genes of interest.
Automatically generated - may contain errors
4 protocols using pgl3 basic luciferase vector
1
Validating miR-26a binding to SLC7A11 mRNA
2
Luciferase Reporter Assay for circZFR and ZEB2
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For luciferase reporter assay, circZFR-WT, circZFR-mutant, ZEB2-WT and ZEB2-mutant were constructed into pGL3-Basic luciferase vector (GenePharma, Shanghai, China) to construct reporter plasmids. Afterwards, cells were co-transfected with indicated reporter plasmids and microRNA using Lipofectamine 2000 (Life Technologies). Subsequently, the relative luciferase activity was detected by the dual-luciferase reporter assay system (Promega, Madison, WI, U.S.A.) at 48 h post-transfection. Firefly luciferase activity was normalized to renilla luciferase activity.
3
Luciferase Reporter Assay for miR-143-3p
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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)].
4
Luciferase Reporter Assay for miR-143-3p
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MyD88 wild-type and its corresponding mutant were built into pGL3-basic luciferase vector (2 µg/mL; Shanghai GenePharma Co., Ltd., China) to generate the luciferase reporter plasmids. Cells at the third passage were inoculated into 24-well plates at a density of 4×10 4 cells per well and cultured at 37℃. When reached about 70% con uence, cells were co-transfected with luciferase reporter plasmids, miR-143-3p mimic or NC mimic by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scienti c, USA) according to the operating instructions. Luciferase activity was examined with a dual-luciferase reporter assay kit (cat. no. E1910; Promega Corporation, Madison, USA) following transfection for 24 h.
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