Detection of telomerase activity in cultured cells and tumor tissue of lung carcinoma by modified telomeric repeat amplification protocol

Abstract

Telomerase activity is found in various cell types including stem cells, neoplastic cells, and immortalized cells, suggesting a close association with their proliferation capacity. The telomeric repeat amplification protocol (TRAP) has been traditionally used to detect semi-quantitatively the telomerase activity by polyacrylamide gel electrophoresis (PAGE), which is difficult to apply for large scale analysis because of laborious post-PCR manipulation and potential carryover contamination. In the present study, a specific reverse primer was designed and the TRAP protocol was adapted to either PAGE or real-time PCR assay. Using cultured cell lines, the real-time TRAP showed a dramatic improvement in the reliability and accuracy of quantitation of telomerase activity and was able to discriminate the A549 cells from hundreds-fold human embryonic lung cells. Using clinical samples of 60 lung cancers and 8 inflammatory lesions, the real-time TRAP was also superior in quantitation, high-throughput capability and standardization. Our modified real-time TRAP should be applicable for the detection of telomerase activity for the initial screening and progression monitoring of lung cancer patients. Our approach is particularly useful when only limited clinical specimen is available, such as fine needle aspiration or other cytological specimens that may contain only a small number of tumor cells.

Quantification of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase and Correlations with Telomerase Activity in Lung Cancer

“…Consistent with previous report, the fluorescein-probe-based real-time TRAP assay gave more rapid, accurate results for telomerase activity quantification than traditional TRAP with gel electrophoresis [17]. In our previous study, we examined the telomerase activity of 60 lung cancer tissues using SYBR Green real-time TRAP, a relative quantitative analysis, and only found the difference in telomerase activity between NSCLC and CSS [9]. In this study, we applied the RPP-based real-time TRAP to a larger number of lung tumor tissue samples (165 cases) and used standard curve analysis.…”

“…The CHAPS lysis buffer was prepared as described previously [9]. The cell pellets were resuspended in ice-cold lysis buffer (5000 cells/ µL) and incubated for 30 min on ice.…”

Telomerase activity associated with progression of cervical lesions in a group of Colombian patients

“…Telomerase activity can be measured in vitro by using the telomeric repeat amplification protocol 16 . This assay has been used to test telomerase activity in numerous cancer specimens, and in uncultured and cultured samples of normal tissue from many cell types 16 .…”

Gastrokine 1 induces senescence and apoptosis through regulating telomere length in gastric cancer

“…The kit also included a telomerase substrate oligonucleotide (TSR) control template, providing a standard curve and a positive control. Telomerase activity was evaluated using the relative quantities according to an internal calibrator using the 2- ΔΔ CT method [ 42 ].…”