Telomerase activity detection by TRAP-ELISA

SKM-1 cell telomerase activity was assessed using the TEELISA kit (cat. no. CSB-E08021h; Cusabio Technology LLC, USA) according to the manufacturer's instructions. Following treatment for 24 h, cells (1x10⁶) were centrifuged at 4200 x g for 20 min at 4˚C. The cell pellet was incubated with cold lysis buffer (200 µl) form the aforementioned kit, for 30 min on ice. The supernatant (2 µl) was used as the TRAP reaction template. Subsequently, 25 µl reaction mixture was added to the PCR reaction tube and the total volume was adjusted to 50 µl with DEPC-treated sterilized double distilled water to perform the TRAP reaction according to the following protocol: primer extension for 10 min at 25˚C; telomerase inactivation for 5 min at 94˚C; 30 cycles of denaturation for 30 sec at 94˚C, annealing for 30 sec at 50˚C and extension for 90 sec at 72˚C; and extension for 10 min at 72˚C. The amplified product (5 µl) and the denaturant (20 µl) were mixed and maintained at room temperature for 10 min. Subsequently, 225 µl hybridization solution (containing digoxin-labeled probe) was added, and after mixing, 100 µl mixture was added to an anti-biotin coated plate and incubated for 2 h at 37˚C (300 rpm). Then peroxidase was added and developed with TMB substrate for 30 min at room temperature. Finally, 100 µl stop solution was added. The A value of each well (detection wavelength, 450 nm; reference wavelength, 690 nm) and the magnitude of the A value represented the level of telomerase activity.