title: SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity.
abstract: 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.
authors.affiliation: Transplant Research Institute, University of California, Davis Medical Center, 4635 Second Avenue, Suite 1001, Sacramento, CA 95817, USA. hwege@ucdavis.edu
authors.forename: Henning
authors.initials: H
authors.lastname: Wege
authors.forename: Michael S
authors.initials: MS
authors.lastname: Chui
authors.forename: Hai T
authors.initials: HT
authors.lastname: Le
authors.forename: Julie M
authors.initials: JM
authors.lastname: Tran
authors.forename: Mark A
authors.initials: MA
authors.lastname: Zern
mesh_terms: D052160:Benzothiazoles; D002460:Cell Line; D002478:Cells, Cultured; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011804:Quinolines; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000201:enzymology
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D019098:Telomerase