Trapeze kit

Unless otherwise specified, telomerase activity was analyzed using the TRAPeze kit (Millipore) per manufacturer's directions. The telomeric extension products were separated by 10% TBE-PAGE and visualized by phosphorimaging (Molecular Dynamics).
For 47A-hTER or WT-hTER specific TRAP assay, TRAP reaction was carried out as described in [59 (link)] except that the return primer 5’-GCGCGGTACCCATACCCATACCCAAACCCA-3’ was used to detect 47A-hTER activity, and the return primer 5’- GCGCGGTACCCTTACCCT TACCCTAACCCT-3’ was used to detect WT-hTER activity. TRAP products intensity in each lane were quantified by the ImageQuant Software and normalized to the respective internal control intensity.

Telomerase activity was determined using the TRAPeze telomerase detection kit (Millipore, Burlington, MA, USA), following the manufacturer’s protocol. In brief, frozen WT xenograft tissues were homogenized in CHAPS buffer and incubated at 25 °C for 40 min. For negative controls, lysates were heated up to 95 °C for 5 min to deactivate telomerase. Nucleotide templates for deposition of telomeric repeats and primers provided in the kit were added. The reaction was subjected to 29 PCR cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s. TRAP assay products were run on a 15% polyacrylamide gel and stained with SYBR green (ThermoFisher) for visual representation of telomerase activity and confirmation of positive and negative controls. Heat treatment (which inactivates telomerase) served as a negative control for each specimen. The human telomerase-positive control cell pellet provided in the TRAPeze kit (Millipore) served as a positive control for telomerase activity. TRAP assay products were quantified by capillary electrophoresis using 5′-fluorescent labeled (5′ 6-FAM) primers (IDT, Coralville, IA, USA) and calculating the total product generated (TPG). One TPG corresponds to the amount of telomerase activity detected in one immortal cell.