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Robust measurement of telomere length in single cells
Friday, 2013/05/24 | 07:43:08

Fang Wanga,b, Xinghua Panc,1, Keri Kalmbacha, Michelle L. Seth-Smitha, Xiaoying Yeb, Danielle M. F. Antumesa,d,e, Yu Yinb, Lin Liub,1, David L. Keefea,1, and Sherman M. Weissmanc,1

 

Author Affiliations

 

1.aDepartment of Obstetrics and Gynecology, New York University Langone Medical Center, NY 10016;
2.bState Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China;
3.cDepartment of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005;
4.dDepartment of Pathology, Fluminense Federal University, 24033-900, Rio de Janeiro, Brazil; and
5.eCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) Foundation, Ministry of Education of Brazil, 70040-020, Brasilia, Brazil

 

  1. Contributed by Sherman M. Weissman, April 9, 2013 (sent for review February 5, 2013)

 

Significance

 

Telomeres are the structures at the ends of chromosomes that protect these ends from degradation or joining to one another. Telomeres consist of repeat DNA sequences and the length is gradually eroded as the cell ages. The ability to measure telomere length in individual cells would be important for studies of cell senescence, malignancy, stem cell renewal, and human fertility. We have developed a robust and practical method for estimating the telomere length of single cells, and used this method to demonstrate the heterogeneity or changes of telomere length in several systems.

 

Abstract

 

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

 

http://www.pnas.org/content/110/21/E1906.abstract.html?etoc

PNAS May 21, 2013 vol. 110 no. 21 E1906-E1912

 

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