Dual glo stop glo

HEK293T cells were seeded in DMEM (10% FBS, no antibiotics) in a six-well plate and incubated overnight. After ∼24 h, transfection with plasmid DNA and siRNA was performed. Cells were transfected with 2 μg of del4 wildtype or mutated plasmid, 0.5 μg pRL-TK Renilla plasmid and a 1:1:1:1 mixture of siRNA against TOP1 (HS TOP1 #3510923 GeneSolution Qiagen), TOP2α (HS TOP2α #342786 GeneSolution Qiagen), TOP2β (HS TOP2β #3389257GeneSolution Qiagen) or scrambled siRNA (AllStars Negative Control siRNA #1027281, Qiagen). 72 h after transfection, dead cells and cell debris were removed by two times wash with PBS before the remaining cells were trypsinated. 4000 transfected cells were seeded in a 96-well plate in 50 μl DMEM (10% FBS, no antibiotics). 50 μl Dual-Glo Luciferase Reagent (Promega) was added and the cells were incubated 10 min at room temperature before the firefly luminescence of the cells was measured using Fluostar Optima (BMG Labtech) with settings on 0.2 s positioning delay, 0.0 s measurement start time and 0.1 s as the interval. This was followed by addition of 50 μl Dual-Glo Stop & Glo (Promega), and the cells were left for additional 10 min of incubation at room temperature before the renilla luminescence was measured using the same settings.