Luciferase assay kit

Cells were subcultured in 12-well plates at a density 5 x 10⁴ cells/well one day before transfection. To investigate the effect of PIN1 silencing on EPO-HRE reporter activity, cells were transfected with PIN1 siRNA or mock siRNA for 72 h at 37°C. The cells were transfected with plasmids including luciferase-linked reporter gene (EPO-HRE luc) and β-galactosidase (β-gal) expression vector. After 24 h transfection, cells were lysed, and luciferase activities were measured. The β-gal activity was used to normalize transfection efficiency.
Bioluminescence image was obtained using a luciferase assay kit (Applied Biosystems, Carlsbad, CA, USA). The plates were washed twice with PBS, and then lysis solution was added to each well. Cell lysates were transferred to a 96 well microplate and luciferase activities were measured by a Wallac 1420 VICTOR3 V (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) luminometer.

Luciferase assays were performed on 293T cells using the pMIR-REPORT System (Applied Biosystems, USA) following the manufacturer's protocol. Briefly, either the pMIR-REPORT-ZFX-WT-3′UTR plasmid or a mutant form of the plasmid (3 μg) was co-transfected with a pMIR-REPORT β-gal control plasmid (1 μg) into 293T cells (10⁶) with or without miR-101 overexpression using 15 mL of Lipofectamine 2000 (Invitrogen). Twenty-four hours later, luciferase activity was measured using a Luciferase Assay Kit (Applied Biosystems). To accomplish this, lysis solution (250 mL) was added to the cells, and the cells were detached from the plate with a cell scraper. The cell lysate was then transferred into a microfuge tube and centrifuged at 4°C for 5 min. The resulting supernatant was transferred into a fresh tube, and 50 mL of cell extract was transferred into a luminometer tube. Following this, 100 mL of Substrate A (ATP solution) and 100 mL of Substrate B (luciferin solution) were added. Following a 2-s pre-measurement delay, a luminometer was used to measure light emission from the tube for 10 s. β-galactosidase activity was tested using a β-Gal Assay Kit (Invitrogen) following the manufacturer's instructions. Relative luciferase activity was obtained by normalizing luciferase expression against β-gal expression.