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.

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“…Telomerase (TERT) activity assay Quantitative determination of telomerase activity was performed using a validated quantitative telomerase detection kit (Allied Biotech, Inc., Ijamsville, MD, USA) (high-sensitive real-time quantitative Telomeric Repeat Amplification Protocol) according to the manufacturer's protocol (Wege et al 2003 …”

Infectivity enhanced, hTERT promoter-based conditionally replicative adenoviruses are useful for SCLC treatment

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Quantitative telomerase activity in circulating human leukocytes: utility of real-time telomeric repeats amplification protocol (RQ-TRAP) in a clinical/epidemiological setting

“…A serial dilutioncalibration curve was also performed on each plate to further minimize inter-plate variability, as recommended by the manufacturer. Each sample was analyzed in duplicate as described previously (9). Melting curve analysis was performed on each run to verify specificity and identity of the PCR products.…”

“…Recently, a real-time quantitative SYBR Green highsensitivity telomeric repeat amplification protocol (RQ-TRAP) for rapid quantitation of telomerase activity has been developed (8,9). This procedure may serve as an alternative for the study of telomerase regulation and its relevance to somatic cell physiology, particularly in population studies.…”

“…This procedure may serve as an alternative for the study of telomerase regulation and its relevance to somatic cell physiology, particularly in population studies. Furthermore, the sensitivity of the RQ-TRAP assay has been demonstrated to be greater than the TRAP-ELISA assay (9). We evaluated the applicability/utility of RQ-TRAP to determine telomerase activity in circulating human leukocytes in blood samples collected in a manner that mimics routine blood collection/storage typically performed for clinical/epidemiological investigations.…”