Detection of hTERT protein by flow cytometry

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of ca ncers and serves to counter the consequences of telomere shortening. The pr otein component, hTERT, is believed to be the catalytic subunit of human te lomerase and its expression at the mRNA level correlates well with telomera se activity in vitro. Current techniques for assaying telomerase activity d etect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows d etection of intranuclear hTERT while maintaining identifiable cell populati on characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differen tiation experiments and in primary cells, a good correlation existed betwee n hTERT expression measured by flow cytometry and telomerase activity detec ted by the telomeric repeat amplification protocol (TRAP). The method devel oped offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investig ations, facilitating the analysis of hTERT expression in mixed cell populat ions.