title: Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow-FISH techniques.
abstract: Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.
authors.affiliation: Center for Human Immunology, Autoimmunity, and Inflammation, National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Taylor
authors.initials: T
authors.lastname: Wand
authors.affiliation: Hematology Branch, National Heart Lung and Blood Institute National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Mike
authors.initials: M
authors.lastname: Fang
authors.affiliation: Hematology Branch, National Heart Lung and Blood Institute National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Christina
authors.initials: C
authors.lastname: Chen
authors.affiliation: Hematology Branch, National Heart Lung and Blood Institute National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Nathan
authors.initials: N
authors.lastname: Hardy
authors.affiliation: Flow Cytometry Core, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: J Philip
authors.initials: JP
authors.lastname: McCoy
authors.affiliation: Hematology Branch, National Heart Lung and Blood Institute National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Dumitriu
authors.affiliation: Center for Human Immunology, Autoimmunity, and Inflammation, National Institutes of Health, Bethesda, Maryland, 28092.|Hematology Branch, National Heart Lung and Blood Institute National Institutes of Health, Bethesda, Maryland, 28092.
authors.forename: Neal S
authors.initials: NS
authors.lastname: Young
authors.affiliation: Center for Human Immunology, Autoimmunity, and Inflammation, National Institutes of Health, Bethesda, Maryland, 28092. biancoa@mail.nih.gov.
authors.forename: Angélique
authors.initials: A
authors.lastname: Biancotto
mesh_terms: D000328:Adult; D000368:Aged; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D020135:Peptide Nucleic Acids / Q000737:chemistry; D011419:Propidium / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D016615:Telomere / Q000737:chemistry*; D055815:Young Adult
chemical_list: D020135:Peptide Nucleic Acids; D011419:Propidium; D004247:DNA; D019793:Fluorescein
keywords: Flow-FISH; TRF2; qPCR; telomere