Cells were transfected using Lipofectamine Plus (Thermofisher) with PSA-luciferase [48 (link)], MMTV-luciferase or PPRE-luciferase, and pRL-SV40 (Promega) as a control. Gene specific or negative control siRNA (Qiagen) was transfected together with the plasmids when applicable. Cells were transferred to a 96-well plate 24 hours after transfection and treated with the appropriate drugs dissolved in media supplemented with charcoal-stripped serum for another 24 hours. Luciferase activity was assayed 24 hours after treatment using the dual-luciferase reporter assay system (Promega). Student’s t-test (two-sided and equal variance) was performed and association was considered significant when p < 0.05 and indicated by an asterisk.
Protocol detail
Dual-Luciferase Assay for Gene Regulation
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After treatment
Assay
Cells
Charcoal
Drugs
Gene
Lipofectamine
Luciferase
Mmtv
Plasmids
Promega
Serum
Sirna
Student
Sv40
Transfection
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Variable analysis
independent variables
- Transfection with PSA-luciferase, MMTV-luciferase or PPRE-luciferase plasmids
- Transfection with gene specific or negative control siRNA
- Luciferase activity
- Transfection with pRL-SV40 plasmid as a control
- Treatment with drugs dissolved in media supplemented with charcoal-stripped serum
- Positive control: pRL-SV40 plasmid
- Negative control: Negative control siRNA
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