Analytical Validation of Telomerase Activity for Cancer Early Detection: TRAP/PCR-CE and hTERT mRNA Quantification Assay for High-Throughput Screening of Tumor Cells
J P. Jakupciak, Wenhua Wang, Peter E. Barker, S Srivastava, Donald H. Atha
Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme posses aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as TRAP/PCR or antibody-based RIA are low throughput and not robust enough to easily accommodate the required statistical analysis to determine if telomerase activity is a practical biomarker. As part of the National Cancer Institute Early Detection Research Network of analytical validation, we have developed a robot assisted TRAP assay (RApidTRAP) of telomerase, a potential biomarker for cancer early detection. Measurements of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA were performed in conjunction with measurement of telomerase activity. For this purpose we determined h TERT mRNA concentration and telomerase activity in human normal (RPE-wi) and cancer (A549) cell lines as well as in human serum (SRM 1951A). Telomerase activity measurements were made using the TRAP/PCR CE method on 50 to 500 cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using the TRAP/PCR CE method on 50 to 500 cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using specific primers and probes on a LightCycler in the range of 10 to 7000 cells/reaction. Comparison of high-throughput telomerase activity measurements using the robot and those performed manually were consistent in sensitivity and reproducibility. Using this combination of telomerase activity and hTERT mRNA measurements, the automated system improved efficiency and accuracy over traditional TRAP/PCR methods.
, Wang, W.
, Barker, P.
, Srivastava, S.
and Atha, D.
Analytical Validation of Telomerase Activity for Cancer Early Detection: TRAP/PCR-CE and hTERT mRNA Quantification Assay for High-Throughput Screening of Tumor Cells, Journal of Molecular Diagnostics
(Accessed December 1, 2023)