Assessment of telomere length in hematopoietic interphase cells using in situ hybridization and digital fluorescence microscopy

Telomeres are G/C‐rich repetitive DNA sequences at the end of all eukaryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescence. The standard procedure for measurement of telomere length is Southern blot (SB) hybrid...

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Bibliographic Details
Published in:Cytometry (New York, N.Y.) N.Y.), 1998-07, Vol.32 (3), p.163-169
Main Authors: De Pauw, E. S. D., Verwoerd, N. P., Duinkerken, N., Willemze, R., Raap, A. K., Fibbe, W. E., Tanke, H. J.
Format: Article
Language:English
Subjects:
Adult
Cell Nucleus - genetics
Cells, Cultured
Chromosomes, Human - genetics
digital fluorescence microscopy
Fetus
hematopoietic cells
Hematopoietic Stem Cells - chemistry
Humans
In Situ Hybridization, Fluorescence
Interphase - genetics
interphase cells
Metaphase - genetics
Microscopy, Fluorescence
PNA FISH
Telomere - genetics
telomeric length
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Summary:Telomeres are G/C‐rich repetitive DNA sequences at the end of all eukaryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescence. The standard procedure for measurement of telomere length is Southern blot (SB) hybridization with a telomere‐specific probe. However, in using this technique no information can be obtained on variation in telomeric fragments due to interchromosomal, intrachromosomal, and intercellular differences. Lansdorp et al. (Hum Mol Genet 5:685–691, 1996) developed a method to measure individual telomeres, using in situ hybridization on metaphase chromosomes, employing peptide nucleic acid (PNA) probes and digital fluorescence microscopy. In this paper we describe a method that can be used to assess telomeric length in interphase cells. An algorithm was developed to measure the total intranuclear fluorescence in situ hybridization (FISH) signal, which features accurate correction for the local autofluorescence. Application of this methodology to samples of fetal liver, umbilical cord blood, and adult bone marrow cells showed a gradual decrease of average telomeric length. Southern blot analysis and PNA FISH measurements on chromosomes in the same samples showed similar results. Advantages of interphase measurements include the possibility of studying nonproliferating cells, thus avoiding selection and cell culturing. Cytometry 32:163–169, 1998. © 1998 Wiley‐Liss, Inc.
ISSN:0196-4763
1097-0320
DOI:10.1002/(SICI)1097-0320(19980701)32:3<163::AID-CYTO1>3.0.CO;2-L