Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.
Bottom Line: 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.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.
Affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.Show MeSH
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Mentions: There are sequence differences in the telomerase RNA component between human and mouse TERC (26). We examined whether ddTRAP, similar to gel based TRAP, was able to detect mouse telomerase activity using immortal NIH3T3 mouse fibroblasts (Figure 7). The magnitude of the difference of telomerase activity between TRAP and ddTRAP was similar in HeLa compared to NIH3T3 cells (Figure 7). Optimization of telomerase substrates that are more compatible with the mouse telomerase RNA component will be needed for determination of absolute molecule counts of mouse telomerase activity. The present data does not address how efficiently mouse telomerase works on the TS primer, but ddTRAP offers a suitable quantitative surrogate to traditional gel-based methods.
Affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.