Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH

Nature Protocols volume 12, pages 1855–1870 (2017)Cite this article

Subjects

Abstract

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA)3 peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 3: Telomere analysis.
Figure 2: The global steps required for CQ-FISH.

Similar content being viewed by others

References

  1. Blackburn, E.H. Switching and signaling at the telomere. Cell 106, 661–673 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Wong, J.M. & Collins, K. Telomere maintenance and disease. Lancet 362, 983–988 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Cawthon, R.M., Smith, K.R., O'Brien, E., Sivatchenko, A. & Kerber, R.A. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet 361, 393–395 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Epel, E.S. et al. Accelerated telomere shortening in response to life stress. Proc. Natl. Acad. Sci. USA 101, 17312–17315 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Oh, H. et al. Telomere attrition and Chk2 activation in human heart failure. Proc. Natl. Acad. Sci. USA 100, 5378–5383 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Raymond, A.R., Norton, G.R., Sareli, P., Woodiwiss, A.J. & Brooksbank, R.L. Relationship between average leucocyte telomere length and the presence or severity of idiopathic dilated cardiomyopathy in black Africans. Eur. J. Heart Fail. 15, 54–60 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Telomeres Mendelian Randomization Collaboration et al. Association between telomere length and risk of cancer and non-neoplastic diseases: a Mendelian randomization study. JAMA Oncol. 3, 636–651 (2017).

  8. Li, C. et al. Relationship between the TERT, TNIP1 and OBFC1 genetic polymorphisms and susceptibility to colorectal cancer in Chinese Han population. Oncotargethttp://dx.doi.org/10.18632/oncotarget. 18378 (2017).

  9. Mourkioti, F. et al. Role of telomere dysfunction in cardiac failure in Duchenne muscular dystrophy. Nat. Cell Biol. 15, 895–904 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Takubo, K. et al. Telomere lengths are characteristic in each human individual. Exp. Gerontol. 37, 523–531 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. van der Harst, P. et al. Telomere length of circulating leukocytes is decreased in patients with chronic heart failure. J. Am. Coll. Cardiol. 49, 1459–1464 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Allsopp, R.C. et al. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10114–10118 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Aida, J. et al. Basal cells have longest telomeres measured by tissue Q-FISH method in lingual epithelium. Exp. Gerontol. 43, 833–839 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Blackburn, E.H., Epel, E.S. & Lin, J. Human telomere biology: a contributory and interactive factor in aging, disease risks, and protection. Science 350, 1193–1198 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Heaphy, C.M. et al. Altered telomeres in tumors with ATRX and DAXX mutations. Science 333, 425 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hurwitz, L.M. et al. Telomere length as a risk factor for hereditary prostate cancer. Prostate 74, 359–364 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Klewes, L. et al. Three-dimensional nuclear telomere organization in multiple myeloma. Transl. Oncol. 6, 749–756 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Knecht, H., Sawan, B., Lichtensztejn, Z., Lichtensztejn, D. & Mai, S. 3D telomere FISH defines LMP1-expressing Reed-Sternberg cells as end-stage cells with telomere-poor 'ghost' nuclei and very short telomeres. Lab. Invest. 90, 611–619 (2010).

    Article  PubMed  Google Scholar 

  19. Meeker, A.K. et al. Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining. Am. J. Pathol. 160, 1259–1268 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Meeker, A.K. et al. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin. Cancer Res. 10, 3317–3326 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Meeker, A.K. et al. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62, 6405–6409 (2002).

    CAS  PubMed  Google Scholar 

  22. Montgomery, E., Argani, P., Hicks, J.L., DeMarzo, A.M. & Meeker, A.K. Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically. Am. J. Pathol. 164, 1523–1529 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Plentz, R.R. et al. Telomere shortening and inactivation of cell cycle checkpoints characterize human hepatocarcinogenesis. Hepatology 45, 968–976 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Sasaki, M., Ikeda, H., Yamaguchi, J., Nakada, S. & Nakanuma, Y. Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence. Hepatology 48, 186–195 (2008).

    Article  PubMed  Google Scholar 

  25. Shekhani, M.T. et al. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors. Hum. Pathol. 54, 106–112 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. van Heek, N.T. et al. Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia. Am. J. Pathol. 161, 1541–1547 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Buchardt, O., Egholm, M., Berg, R.H. & Nielsen, P.E. Peptide nucleic acids and their potential applications in biotechnology. Trends Biotechnol. 11, 384–386 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. de Lange, T. et al. Structure and variability of human chromosome ends. Mol. Cell. Biol. 10, 518–527 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Gardner, M. et al. Gender and telomere length: systematic review and meta-analysis. Exp. Gerontol. 51, 15–27 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Hande, M.P., Samper, E., Lansdorp, P. & Blasco, M.A. Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. J. Cell Biol. 144, 589–601 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Harley, C.B., Futcher, A.B. & Greider, C.W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990).

    Article  CAS  PubMed  Google Scholar 

  32. Kimura, M. et al. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nat. Protoc. 5, 1596–1607 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Cawthon, R.M. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 37, e21 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Ding, C. & Cantor, C.R. Quantitative analysis of nucleic acids—the last few years of progress. J. Biochem. Mol. Biol. 37, 1–10 (2004).

    CAS  PubMed  Google Scholar 

  35. O'Callaghan, N.J. & Fenech, M. A quantitative PCR method for measuring absolute telomere length. Biol. Proced. Online 13, 3 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Aviv, A., Valdes, A.M. & Spector, T.D. Human telomere biology: pitfalls of moving from the laboratory to epidemiology. Int. J. Epidemiol. 35, 1424–1429 (2006).

    Article  PubMed  Google Scholar 

  37. Liehr, T. Fluorescence In Situ Hybridization (FISH)—Application Guide, 2nd edn. XVIII, 451 (2009).

    Google Scholar 

  38. Henderson, S., Allsopp, R., Spector, D., Wang, S.S. & Harley, C. In situ analysis of changes in telomere size during replicative aging and cell transformation. J. Cell Biol. 134, 1–12 (1996).

    Article  CAS  PubMed  Google Scholar 

  39. Lansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996).

    Article  CAS  PubMed  Google Scholar 

  40. Martens, U.M. et al. Short telomeres on human chromosome 17p. Nat. Genet. 18, 76–80 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Zijlmans, J.M. et al. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. USA 94, 7423–7428 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Liao, H.S. et al. Cardiac-specific overexpression of cyclin-dependent kinase 2 increases smaller mononuclear cardiomyocytes. Circ. Res. 88, 443–450 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Baerlocher, G.M., Vulto, I., de Jong, G. & Lansdorp, P.M. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat. Protoc. 1, 2365–2376 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Hultdin, M. et al. Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res. 26, 3651–3656 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Poon, S.S., Martens, U.M., Ward, R.K. & Lansdorp, P.M. Telomere length measurements using digital fluorescence microscopy. Cytometry 36, 267–278 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. Rufer, N. et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J. Exp. Med. 190, 157–167 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E. & Lansdorp, P.M. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat. Biotechnol. 16, 743–747 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. Soor, G.S. et al. Hypertrophic cardiomyopathy: current understanding and treatment objectives. J. Clin. Pathol. 62, 226–235 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Briceno, N., Schuster, A., Lumley, M. & Perera, D. Ischaemic cardiomyopathy: pathophysiology, assessment and the role of revascularisation. Heart 102, 397–406 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Luk, A., Ahn, E., Soor, G.S. & Butany, J. Dilated cardiomyopathy: a review. J. Clin. Pathol. 62, 219–225 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. Parrillo, J.E. Inflammatory cardiomyopathy (myocarditis): which patients should be treated with anti-inflammatory therapy? Circulation 104, 4–6 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Boudina, S. & Abel, E.D. Diabetic cardiomyopathy, causes and effects. Rev. Endocr. Metab. Disord. 11, 31–39 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Camacho, P., Fan, H., Liu, Z. & He, J.Q. Small mammalian animal models of heart disease. Am. J. Cardiovasc. Dis. 6, 70–80 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Sironi, A.M. et al. Impact of increased visceral and cardiac fat on cardiometabolic risk and disease. Diabet. Med. 29, 622–627 (2012).

    Article  CAS  PubMed  Google Scholar 

  55. Williams, Y. et al. Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots. J. Microsc. 232, 91–98 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. le Maire, M., Champeil, P. & Moller, J.V. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508, 86–111 (2000).

    Article  CAS  PubMed  Google Scholar 

  57. Flores, I. et al. The longest telomeres: a general signature of adult stem cell compartments. Genes Dev. 22, 654–667 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Araki, T. et al. Mouse model of Noonan syndrome reveals cell type– and gene dosage–dependent effects of Ptpn11 mutation. Nat. Med. 10, 849–857 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Chuang, T.C. et al. The three-dimensional organization of telomeres in the nucleus of mammalian cells. BMC Biol. 2, 12 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Vermolen, B.J. et al. Characterizing the three-dimensional organization of telomeres. Cytometry A 67, 144–150 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. De Marzo and J. Morgan at the Johns Hopkins University for telometer software development. This work was supported by startup funds from University of Pennsylvania and a Pilot and Feasibility Grant from the US National Institutes of Health (P30 AR069619) to F.M.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Maryam Sharifi-Sanjani & Foteini Mourkioti

  2. Departments of Pathology, Oncology and Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Alan K Meeker

  3. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Foteini Mourkioti

  4. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Foteini Mourkioti

Authors
  1. Maryam Sharifi-Sanjani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alan K Meeker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Foteini Mourkioti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S.-S. performed the experiments, troubleshot cardiac experiments, analyzed the data and wrote/edited the manuscript; A.K.M. pioneered in situ hybridization of telomeres coupled with immunofluorescence in human testis, provided expertise, helped develop the automated software for telomere analysis and edited the manuscript; and F.M. conceived the idea to optimize the protocol for cardiac tissues, troubleshot cardiac experiments, wrote/edited the manuscript and provided funds to complete described work. All authors interpreted protocol steps and data.

Corresponding author

Correspondence to Foteini Mourkioti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Protocol optimization.

(A) Cryosections (left) exhibit poor staining unsuitable for telomere quantification. Paraffin cardiac sections (middle and right) maintain a better cardiac morphology with measurable telomere staining. Telomeric probe is shown in red and DAPI for nuclei is shown in blue. Inserts (white rectangles) represent a close up of one nucleus. Telomere staining in paraffin sections is measurable by the telometer software. Note that human cardiac tissues have often increased background autofluorescence, probably due to lipofuscin, also known as “age pigments” and/or red blood cells that are evident at the wavelength of Cy3 detection). However, this is not a concern since this type of autofluorescence (yellow arrowheads) is either cytoplasmic or outside the nucleus and therefore do not interfere with the cardiac telomere assay (see also Figure 2), (B) Representative images of telomere staining coupled with different cardiac markers. Note that the cardiac Troponin T (cTnT) staining (right) is optimal for CQ-FISH, while cardiac Troponin C (cTnC) (middle) is suboptimal and α-actinin (left) is incompatible, (C) Cardiac immunofluorescence after PNA hybridization (right) enhances cardiac staining when compared with the reverse order of staining (left), (D) Heat treatment (>80°C) of cardiac sections should be avoided as they result in high levels of background autofluorescence. Scale bars, 10μm.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sharifi-Sanjani, M., Meeker, A. & Mourkioti, F. Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH. Nat Protoc 12, 1855–1870 (2017). https://doi.org/10.1038/nprot.2017.082

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.082

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing