Wild-type and mutated c-MYB 3′ UTRs were synthesized and inserted into a p-MIR reporter plasmid (Genepharma, Shanghai, China). For luciferase reporter assays, 2 mg of firefly luciferase reporter plasmid, 2 mg of β-gal expression vector (Ambion), and equal amounts (200 pmol) of mimics, inhibitors, or scrambled negative control RNA were transfected into cells. The β-gal vector was used as a transfection control. 24 h after transfection, cells were assayed using a luciferase assay kit (Promega).
β gal expression vector
Manufactured by Thermo Fisher Scientific
The β-gal expression vector is a plasmid used for the expression of the lacZ gene, which encodes the β-galactosidase enzyme. This vector is commonly used as a reporter system in genetic and molecular biology experiments.
Automatically generated - may contain errors
2 protocols using β gal expression vector
1
Luciferase Assay for c-MYB 3'UTR Function
2
miR-34a Regulation of Bcl-2 3'UTR
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The 3′UTR of Bcl-2 containing the predicted target sequence was cloned and inserted into pMIR-REPORTTM-Luciferase vector (Ambion, Austin, TX, USA). Forward primer: 5′-CATGCCTGCCCCAAACAAATA-3′; reverse primer: 5′-AGGGCATTTTTCCCATCGCT-3′. Mutated vector was generated in Invitrogen by replacing the predicted target region with its reverse sequence (from ACUGCC to TGACGG). SH-SY5Y cells were seeded in 24 well plates for 12 h. Afterwards, 0.2 μg of firefly luciferase reporter plasmid, 0.2 μg of β-galactosidase (β-gal) expression vector (Ambion), and 50 nmol/L of miR-34a mimics or scrambled control were transfected into cells. Cells were harvested for luciferase assay (Promega, Madison, WI, USA) 24 h later, and luciferase activities were normalized to β-gal activities.
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