Nucleic Acids Research, 2002, Vol. 31, No. 2 e3
© 2002 Oxford University Press
SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity
Transplant Research Institute, University of California, Davis Medical Center, 4635 Second Avenue, Suite 1001, Sacramento, CA 95817, USA
*To whom correspondence should be addressed. Tel: +1 916 734 7901; Fax: +1 916 734 8097; Email: hwege{at}ucdavis.edu
The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.