The strict suppression of telomerase activity (TA) in terminally differentiated human cells causes a shortening of the chromosome ends after each cell division. This tumor suppression surveillance mechanism is associated with a limited number of cell divisions known as Hayflick limit. Here we present an optimized protocol for measuring TA that combines a fluorescently labeled bait primer and polymerase chain reaction (PCR) amplification with analytical capillary electrophoresis (CE) to achieve a detection limit of one telomerase-positive cell per ten thousand negative cells. In research laboratories today, a broad panel of TRAP assay protocols enables the assessment of the immortality of newly generated cell lines or the unambiguous evaluation of the reprogramming of induced pluripotent stem cells (iPSCs). The present f-TRAP protocol, optimized for routine use, enables fast ad hoc application for single measurements up to a high throughput of mass samples using a triplicate approach of different lysate concentrations. Final CE analysis facilitates standardized data processing and storage for documentation of results and could make f-TRAP a useful assay in research and clinical oncology.
Keywords: capillary electrophoresis; fluorescent telomeric repeat amplification protocol (f-TRAP; human telomerase reverse transcriptase (hTERT); immortalization; non-radioactive; telomerase activity (TA).
Telomerase activity is the most universal of all known tumor markers and a promising target for anti-tumor therapies. The robustness and high sensitivity of f-TRAP can (i) demonstrate early validation of cell line immortality in the establishment of model systems, (ii) lead to rapid detection of successful reprogramming or differentiation of cells in vitro and (iii) be used in high throughput assays in the search for new inhibitors of telomerase activity.