title: Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures.
abstract: Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β- variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.
authors.affiliation: UCSF/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Novak
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Hart
authors.affiliation: UCSF/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA Department of Chemistry, University of California, Berkeley, CA 94720, USA ramathies@berkeley.edu.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D003474:Curcumin / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D012313:RNA / Q000032:analysis; D012326:RNA Splicing / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059010:Single-Cell Analysis; D019098:Telomerase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; D003474:Curcumin