Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity

Background: Telomerase is a ribonucleoprotein enzyme associated with immort alization and transformation of human cells. The telomeric repeat amplifica tion protocol (TRAP) is widely used for the detection of telomerase activit y. The TRAP method, although highly sensitive and specific because it inclu des PCR amplification, is laborious and does not provide precise quantitati ve information. Methods: We developed a real-time quantitative TRAP (RTQ-TRAP) system by co mbining a real-time PCR technique with the conventional TRAP method. Telome rase activity in human tumor cell lines and in 13 lymphoma samples was meas ured using the RTQ-TRAP assay, and the results obtained from the samples us ing the RTQ-TRAP method were compared with the conventional TRAP method. Results: The RTQ-TRAP method was both accurate and reproducible in measurin g telomerase activity in a dilution series of protein extracts from HL60 ce lls. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-T RAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as deter mined using RTQ-TRAP, was much shorter than the half-life reported previous ly. Conclusions: Our results suggest that the conventional TRAP assay frequ ently overestimates telomerase activity in tumor samples. The RTQ-TRAP meth od is thus a useful tool to rapidly and precisely quantify telomerase activ ity. (C) 2001 American Association for Clinical Chemistry.