Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

Andrew T. Ludlow, Jerome D. Robin, Mohammed Sayed, Claudia M. Litterst, Dawne N. Shelton, Jerry W. Shay, Woodring E. Wright

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

Original languageEnglish (US)
JournalNucleic Acids Research
Volume42
Issue number13
DOIs
StatePublished - 2014

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Telomerase
Polymerase Chain Reaction
Enzymes
Telomere
Cell Line
Mitogens
HeLa Cells
Electrophoresis
Blood Cells
Gels
Technology
DNA

ASJC Scopus subject areas

  • Genetics

Cite this

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. / Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

In: Nucleic Acids Research, Vol. 42, No. 13, 2014.

Research output: Contribution to journalArticle

Ludlow, Andrew T. ; Robin, Jerome D. ; Sayed, Mohammed ; Litterst, Claudia M. ; Shelton, Dawne N. ; Shay, Jerry W. ; Wright, Woodring E. / Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 13.
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