Telomeric repeat amplification protocol (TRAP) assay

Trap assay was performed using the TRAPeze® Telomerase Detection Kit (S7700-KIT; Millipore). Briefly, fresh skin tissue was obtained and floated with the dermis side down in 0.25% trypsin at 4°C O/N. On the next day, the dermis was peeled off and the epidermis was homogenized while kept on ice. CHAPS lysis buffer was added (200μl per 10μg of tissue) and samples were incubated on ice for 30 min. Then, 160μL of the supernatant was collected, and the protein concentration was determined. A reaction mix containing TRAP buffer (20 mM Tris–HCl, pH 8.3, 1.5 mM MgCl₂, 63 mM KCl, 0.05% (v/v) Tween-20, 1 mM EDTA, and 0.01% BSA; TRAPeze telomerase detection kit), supplemented with dNTP mix, TS primer, TRAP primer mix, dH₂O, Taq polymerase at indicated concentrations was prepared and incubated with the samples at 30°C for 30 min. PCR was used for amplication (94°C for 30 s, 59°C for 30 s, 72°C for 1 min for 30 cycles and 72°C for 7 min in a thermocycler. PCR samples were run on a 10% (w/v) native-PAGE gel in 0.5XTBE for 1 h at 150 V. After electrophoresis, the gel was stained with ethidium bromide for 10 min at room temperature. The telomerase activity was estimated by measuring the intensity of the bands using ImageJ software.