Philos Trans R Soc Lond B Biol Sci 373(1741): 20160451

Comparison of telomere length measurement methods

Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA

e-mail: ude.nretsewhtuostu@yahs.yrrej

One contribution of 19 to a theme issue ‘Understanding diversity in telomere dynamics’.

Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA

One contribution of 19 to a theme issue ‘Understanding diversity in telomere dynamics’.

Accepted 2017 Nov 8.

Abstract

The strengths and limitations of the major methods developed to measure telomere lengths (TLs) in cells and tissues are presented in this review. These include Q-PCR (Quantitative Polymerase Chain Reaction), TRF (Terminal Restriction Fragment) analysis, a variety of Q-FISH (Quantitative Fluorescence In Situ Hybridization) methods, STELA (Single TElomere Length Analysis) and TeSLA (Telomere Shortest Length Assay). For each method, we will cover information about validation studies, including reproducibility in independent laboratories, accuracy, reliability and sensitivity for measuring not only the average but also the shortest telomeres. There is substantial evidence that it is the shortest telomeres that trigger DNA damage responses leading to replicative senescence in mammals. However, the most commonly used TL measurement methods generally provide information on average or relative TL, but it is the shortest telomeres that leads to telomere dysfunction (identified by TIF, Telomere dysfunction Induced Foci) and limit cell proliferation in the absence of a telomere maintenance mechanism, such as telomerase. As the length of the shortest telomeres is a key biomarker determining cell fate and the onset of senescence, a new technique (TeSLA) that provides quantitative information about all the shortest telomeres will be highlighted.

This article is part of the theme issue ‘Understanding diversity in telomere dynamics’.

Keywords: Q-PCR, Q-FISH, TRF, STELA, TeSLA

Abstract

Acknowledgements

We thank Drs Pat Monaghan, Dan Nussey and Mark Hausmann for organizing meetings between evolutionary and biomedical researchers and the Leverhulme Trust for the support of these meetings.

Acknowledgements

References

1. Palm W, de Lange T. 2008 How shelterin protects mammalian telomeres. Annu. Rev. Genet.42, 301–334. (10.1146/annurev.genet.41.110306.130350) [] [[PubMed][Google Scholar]
2. Blackburn EH. 2001 Switching and signaling at the telomere. Cell106, 661–673. (10.1016/S0092-8674(01)00492-5) [] [[PubMed][Google Scholar]
3. Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T. 1999 Mammalian telomeres end in a large duplex loop. Cell97, 503–514. (10.1016/S0092-8674(00)80760-6) [] [[PubMed][Google Scholar]
4. de Lange T. 2002 Protection of mammalian telomeres. Oncogene21, 532–540. (10.1038/sj.onc.1205080) [] [[PubMed][Google Scholar]
5. Harley CB, Futcher AB, Greider CW. 1990 Telomeres shorten during ageing of human fibroblasts. Nature345, 458–460. (10.1038/345458a0) [] [[PubMed][Google Scholar]
6. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. 1990 Telomere reduction in human colorectal carcinoma and with ageing. Nature346, 866–868. (10.1038/346866a0) [] [[PubMed][Google Scholar]
7. Lindsey J, McGill NI, Lindsey LA, Green DK, Cooke HJ. 1991 In vivo loss of telomeric repeats with age in humans. Mutat. Res.256, 45–48. (10.1016/0921-8734(91)90032-7) [] [[PubMed][Google Scholar]
8. Shay JW, Wright WE. 1996 The reactivation of telomerase activity in cancer progression. Trends Genet.12, 129–131. (10.1016/0168-9525(96)30018-8) [] [[PubMed][Google Scholar]
9. Fumagalli M, et al. 2012. Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat. Cell Biol.14, 355–365. (10.1038/ncb2466) ] [
10. Hemann MT, Strong MA, Hao LY, Greider CW. 2001 The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell107, 67–77. (10.1016/S0092-8674(01)00504-9) [] [[PubMed][Google Scholar]
11. Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM. 2004 Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol. Cell14, 501–513. (10.1016/S1097-2765(04)00256-4) [] [[PubMed][Google Scholar]
12. von Zglinicki T, Saretzki G, Ladhoff J, d'Adda di Fagagna F, Jackson SP. 2005 Human cell senescence as a DNA damage response. Mech. Ageing Dev.126, 111–117. (10.1016/j.mad.2004.09.034) [] [[PubMed][Google Scholar]
13. Zou Y, Sfeir A, Gryaznov SM, Shay JW, Wright WE. 2004 Does a sentinel or a subset of short telomeres determine replicative senescence?Mol. Biol. Cell15, 3709–3718. (10.1091/mbc.E04-03-0207) ] [[Google Scholar]
14. Kim NW, et al. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science266, 2011–2015. (10.1126/science.7605428) [] [[PubMed]
15. Holohan B, Wright WE, Shay JW. 2014 Cell biology of disease: telomeropathies: an emerging spectrum disorder. J. Cell Biol.205, 289–299. (10.1083/jcb.201401012) ] [[Google Scholar]
16. Townsley DM, Dumitriu B, Young NS. 2014 Bone marrow failure and the telomeropathies. Blood124, 2775–2783. (10.1182/blood-2014-05-526285) ] [[Google Scholar]
17. Opresko PL, Shay JW. 2017 Telomere-associated aging disorders. Ageing Res. Rev.33, 52–66. (10.1016/j.arr.2016.05.009) [] [[PubMed][Google Scholar]
18. Fitzpatrick AL, Kronmal RA, Gardner JP, Psaty BM, Jenny NS, Tracy RP, Walston J, Kimura M, Aviv A. 2007 Leukocyte telomere length and cardiovascular disease in the Cardiovascular Health Study. Am. J. Epidemiol.165, 14–21. (10.1093/aje/kwj346) [] [[PubMed][Google Scholar]
19. Samani NJ, Boultby R, Butler R, Thompson JR, Goodall AH. 2001 Telomere shortening in atherosclerosis. Lancet358, 472–473. (10.1016/S0140-6736(01)05633-1) [] [[PubMed][Google Scholar]
20. Sampson MJ, Hughes DA. 2006 Chromosomal telomere attrition as a mechanism for the increased risk of epithelial cancers and senescent phenotypes in type 2 diabetes. Diabetologia49, 1726–1731. (10.1007/s00125-006-0322-4) [] [[PubMed][Google Scholar]
21. Shay JW. 2016 Role of telomeres and telomerase in aging and cancer. Cancer Discov.6, 584–593. (10.1158/2159-8290.CD-16-0062) ] [[Google Scholar]
22. Nakagawa S, Gemmell NJ, Burke T. 2004 Measuring vertebrate telomeres: applications and limitations. Mol. Ecol.13, 2523–2533. (10.1111/j.1365-294X.2004.02291.x) [] [[PubMed][Google Scholar]
23. Montpetit AJ, et al. 2014. Telomere length: a review of methods for measurement. Nurs. Res.63, 289–299. (10.1097/NNR.0000000000000037) ] [
24. Baird DM. 2005 New developments in telomere length analysis. Exp. Gerontol.40, 363–368. (10.1016/j.exger.2005.02.008) [] [[PubMed][Google Scholar]
25. Vera E, Blasco MA. 2012 Beyond average: potential for measurement of short telomeres. Aging4, 379–392. (10.18632/aging.100462) ] [[Google Scholar]
26. Aubert G, Hills M, Lansdorp PM. 2012 Telomere length measurement—caveats and a critical assessment of the available technologies and tools. Mutat. Res.730, 59–67. (10.1016/j.mrfmmm.2011.04.003) ] [[Google Scholar]
27. Nussey DH, et al. 2014. Measuring telomere length and telomere dynamics in evolutionary biology and ecology. Methods Ecol. Evol.5, 299–310. (10.1111/2041-210X.12161) ] [
28. Cawthon RM. 2002 Telomere measurement by quantitative PCR. Nucleic Acids Res.30, e47 (10.1093/nar/30.10.e47) ] [[Google Scholar]
29. Cawthon RM. 2009 Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res.37, e21 (10.1093/nar/gkn1027) ] [[Google Scholar]
30. Holland AJ, Cleveland DW. 2009 Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis. Nat. Rev.10, 478–487. (10.1038/nrm2718) ] [[Google Scholar]
31. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu JR. 1988 A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc. Natl Acad. Sci. USA85, 6622–6626. (10.1073/pnas.85.18.6622) ] [[Google Scholar]
32. Kimura M, Stone RC, Hunt SC, Skurnick J, Lu X, Cao X, Harley CB, Aviv A. 2010 Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nat. Protoc.5, 1596–1607. (10.1038/nprot.2010.124) [] [[PubMed][Google Scholar]
33. Mender I, Shay JW. 2015 Telomere restriction fragment (TRF) analysis. Bio. Protoc.5, 1658. [Google Scholar]
34. Lansdorp PM, Verwoerd NP, van de Rijke FM, Dragowska V, Little MT, Dirks RW, Raap AK, Tanke HJ. 1996 Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet.5, 685–691. (10.1093/hmg/5.5.685) [] [[PubMed][Google Scholar]
35. Canela A, Vera E, Klatt P, Blasco MA. 2007 High-throughput telomere length quantification by FISH and its application to human population studies. Proc. Natl Acad. Sci. USA104, 5300–5305. (10.1073/pnas.0609367104) ] [[Google Scholar]
36. Der G, Batty GD, Benzeval M, Deary IJ, Green MJ, McGlynn L, McIntyre A, Robertson T, Shiels PG. 2012 Is telomere length a biomarker for aging: cross-sectional evidence from the west of Scotland?PLoS ONE7, e45166 (10.1371/journal.pone.0045166) ] [[Google Scholar]
37. Chen W, Kimura M, Kim S, Cao X, Srinivasan SR, Berenson GS, Kark JD, Aviv A. 2011 Longitudinal versus cross-sectional evaluations of leukocyte telomere length dynamics: age-dependent telomere shortening is the rule. J. Gerontol. A66, 312–319. (10.1093/gerona/glq223) ] [[Google Scholar]
38. Aubert G, Baerlocher GM, Vulto I, Poon SS, Lansdorp PM. 2012 Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes. PLoS Gen.8, e1002696 (10.1371/journal.pgen.1002696) ] [[Google Scholar]
39. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. 2006 Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat. Protoc.1, 2365–2376. (10.1038/nprot.2006.263) [] [[PubMed][Google Scholar]
40. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. 1998 Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat. Biotechnol.16, 743–747. (10.1038/nbt0898-743) [] [[PubMed][Google Scholar]
41. Flores I, Canela A, Vera E, Tejera A, Cotsarelis G, Blasco MA. 2008 The longest telomeres: a general signature of adult stem cell compartments. Genes Dev.22, 654–667. (10.1101/gad.451008) ] [[Google Scholar]
42. Meyne J, et al. 1990. Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma99, 3–10. (10.1007/BF01737283) [] [[PubMed]
43. Verhulst S, Susser E, Factor-Litvak PR, Simons MJ, Benetos A, Steenstrup T, Kark JD, Aviv A. 2015 Commentary: the reliability of telomere length measurements. Int. J. Epidemiol.44, 1683–1686. (10.1093/ije/dyv166) ] [[Google Scholar]
44. Eisenberg DT. 2016 Telomere length measurement validity: the coefficient of variation is invalid and cannot be used to compare quantitative polymerase chain reaction and Southern blot telomere length measurement techniques. Int. J. Epidemiol.45, 1295–1298. (10.1093/ije/dyw191) [] [[PubMed][Google Scholar]
45. Verhulst S, Susser E, Factor-Litvak PR, Simons M, Benetos A, Steenstrup T, Kark JD, Aviv A. 2016 Response to: reliability and validity of telomere length measurements. Int. J. Epidemiol.45, 1298–1301. (10.1093/ije/dyw194) ] [[Google Scholar]
46. Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, Greider CW. 1997 Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell91, 25–34. (10.1016/S0092-8674(01)80006-4) [] [[PubMed][Google Scholar]
47. Erdmann N, Liu Y, Harrington L. 2004 Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc. Natl Acad. Sci. USA101, 6080–6085. (10.1073/pnas.0401580101) ] [[Google Scholar]
48. Gomes NM, Shay JW, Wright WE. 2010 Telomere biology in Metazoa. FEBS Lett.584, 3741–3751. (10.1016/j.febslet.2010.07.031) ] [[Google Scholar]
49. Gomes NMV, et al. 2011. The comparative biology of mammalian telomeres: ancestral states and functional transitions. Aging Cell10, 761–768. (10.1111/j.1474-9726.2011.00718.x) ] [
50. Mender I, Shay JW. 2015 Telomere dysfunction induced foci (TIF) analysis. Bio. Protoc.5, e1656. [Google Scholar]
51. Baird DM, Rowson J, Wynford-Thomas D, Kipling D. 2003 Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat. Genetic.33, 203–207. (10.1038/ng1084) [] [[PubMed][Google Scholar]
52. Lin TT, Letsolo BT, Jones RE, Rowson J, Pratt G, Hewamana S, Fegan C, Pepper C, Baird DM. 2010 Telomere dysfunction and fusion during the progression of chronic lymphocytic leukemia: evidence for a telomere crisis. Blood116, 1899–1907. (10.1182/blood-2010-02-272104) [] [[PubMed][Google Scholar]
53. Sfeir AJ, Chai W, Shay JW, Wright WE. 2005 Telomere-end processing: the terminal nucleotides of human chromosomes. Mol. Cell18, 131–138. (10.1016/j.molcel.2005.02.035) [] [[PubMed][Google Scholar]
54. Zhao Y, Sfeir AJ, Zou Y, Buseman CM, Chow TT, Shay JW, Wright WE. 2009 Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells. Cell138, 463–475. (10.1016/j.cell.2009.05.026) ] [[Google Scholar]
55. Bendix L, Horn PB, Jensen UB, Rubelj I, Kolvraa S. 2010 The load of short telomeres, estimated by a new method, Universal STELA, correlates with number of senescent cells. Aging Cell9, 383–397. (10.1111/j.1474-9726.2010.00568.x) [] [[PubMed][Google Scholar]
56. Lai TP, Wright WE, Shay JW. 2016 Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity. Biotechniques60, 306–309. (10.2144/000114427) [] [[PubMed][Google Scholar]
57. Lai T-P, Zhang N, Noh J, Mender I, Tedone E, Huang E, Wright WE, Danuser G, Shay JW. 2017 A method for measuring the distribution of the shortest telomeres in cells and tissues. Nat. Commun.8, 1356 (10.1038/s41467-017-01291-z) ] [[Google Scholar]
58. Salvador L, Singaravelu G, Harley CB, Flom P, Suram A, Raffaele JM. 2016 A natural product telomerase activator lengthens telomeres in humans: a randomized, double-blind and placebo-controlled study. Rejuv. Res.19, 478–484. (10.1089/rej.2015.1793) ] [[Google Scholar]
59. de Jesus BB, Schneeberger K, Vera E, Tejera A, Harley CB, Blasco MA. 2011 The telomerase activator TA-65 elongates short telomeres and increases health span of adult/old mice without increasing cancer incidence. Aging Cell10, 604–621. (10.1111/j.1474-9726.2011.00700.x) ] [[Google Scholar]
60. Dugdale HL, Richardson DS. 2018 Heritability of telomere variation: it is all about the environment!Phil. Trans. R. Soc. B373, 20160450 (10.1098/rstb.2016.0450) ] [[Google Scholar]
61. Criscuolo F, Smith S, Zahn S, Heidinger BJ, Haussmann MF. 2018 Experimental manipulation of telomere length: does it reveal a corner-stone role for telomerase in the natural variability of individual fitness?Phil. Trans. R. Soc. B373, 20160440 (10.1098/rstb.2016.0440) ] [[Google Scholar]
62. Bateson M, Nettle D. 2018 Why are there associations between telomere length and behaviour?Phil. Trans. R. Soc. B373, 20160438 (10.1098/rstb.2016.0438) ] [[Google Scholar]
63. Baird DM. 2018 Telomeres and genomic evolution. Phil. Trans. R. Soc. B373, 20160437 (10.1098/rstb.2016.0437) ] [[Google Scholar]
64. Young AJ. 2018 The role of telomeres in the mechanisms and evolution of life-history trade-offs and ageing. Phil. Trans. R. Soc. B373, 20160452 (10.1098/rstb.2016.0452) ] [[Google Scholar]
65. Tian X, Doerig K, Park R, Can Ran Qin A, Hwang C, Neary A, Gilbert M, Seluanov A, Gorbunova V. 2018 Evolution of telomere maintenance and tumour suppressor mechanisms across mammals. Phil. Trans. R. Soc. B373, 20160443 (10.1098/rstb.2016.0443) ] [[Google Scholar]
66. Risques RA, Promislow DEL. 2018 All's well that ends well: why large species have short telomeres. Phil. Trans. R. Soc. B373, 20160448 (10.1098/rstb.2016.0448) ] [[Google Scholar]