Abstract
Telomerase is a cellular RNA template-dependent reverse transcriptase that adds telomere repeats to the 3′ ends of chromosomes. Telomerase is expressed almost universally in tumor cells (>85%) to maintain telomere length, thus providing the ability of tumor cells to avoid senescence and to have unlimited replication ability, one of the key hallmarks of cancer. ddTRAP (droplet digital Telomere Repeat Amplification Protocol) is a two-step assay with whole cell lysates that utilizes a telomerase-mediated primer extension followed by droplet digital PCR (ddPCR) detection of extended products. The adoptation of the TRAP assay to ddPCR has resulted in improved throughput, increased sensitivity and better repeatability of the TRAP assay. The protocol described below details our procedures for ddTRAP.
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Acknowledgments
We would like to acknowledge Viresh Patel and George Karlin-Neumann at Bio-Rad for materials support in the development of this technique and critical reading of the manuscript. We also acknowledge Oxford University Press for permissions to use Figure published in our article in Nucleic Acids Research. Ludlow, A.T., Robin, J.D., Sayed, M., Litterst, C.M., Shelton, D.N., Shay, J.W., and Wright, W.E. (2014) Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. Nucleic Acids Res, 42, e104.
Funding
National Institute of Health [AG01228]; National Cancer Institute [CA154805, P50CA70907, T32-CA124334-07, 5P30 CA142543-03]. Funding for open access charge: National Institute of Health Grant [AG01228]. This work was performed in laboratories constructed with support from National Institutes of Health grant C06 RR30414.
Conflict of interest statement
Dawne N. Shelton is a employee of Bio-Rad Laboratories. Bio-Rad Laboratories provided materials and technical support for the development of this technique.
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Ludlow, A.T., Shelton, D., Wright, W.E., Shay, J.W. (2018). ddTRAP: A Method for Sensitive and Precise Quantification of Telomerase Activity. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_29
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_29
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