Quantitative Fluorescence In Situ Hybridization (QFISH) of Telomere Lengths in Tissue and Cells

Jacintha N. O'Sullivan1, Jennifer C. Finley2, Rosa‐ana Risques2, Wen‐Tang Shen2, Katherine A. Gollahon2, Peter S. Rabinovitch2

1 St Vincent's University Hospital, Elm Park, Dublin, 2 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.6
DOI:  10.1002/0471142956.cy1206s33
Online Posting Date:  August, 2005
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Abstract

Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end‐to‐end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ–based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence‐conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin‐fixed paraffin‐embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar‐embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image‐analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.

Keywords: telomere length; quantitative fluorescence in situ hybridization (QFISH); tissue sections; cultured cells; fixation protocols

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Lightly Fixed Tissue for QFISH
  • Alternate Protocol 1: Preparation of Frozen Sections for QFISH
  • Basic Protocol 2: Basic QFISH Staining Procedure
  • Alternate Protocol 2: Modified QFISH for Formalin‐Fixed Archival Tissue
  • Alternate Protocol 3: Preparation of Cultured Cells for QFISH
  • Alternate Protocol 4: Preparation of Cell Pellets Embedded in Paraffin Post‐Culture for QFISH
  • Alternate Protocol 5: Preparation of Epithelial Cells Isolated from Tissue Biopsies for QFISH
  • Basic Protocol 3: Image Analysis to Quantitate Average Telomere Length
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Lightly Fixed Tissue for QFISH

  Materials
  • Tissue, fresh or stored frozen in DMEM containing 10% DMSO
  • Phosphate buffered saline (PBS), pH 7.2 (see recipe)
  • 2% paraformaldehyde (see recipe)
  • 70%, 85%, 90%, 95%, and 100% ethanol
  • Xylene
  • Nitrogen source or liquid nitrogen
  • Microtome
  • Coplin jars
  • Glass microscope slides, charged (e.g., unit 8.5)
  • Heating block for digital adjustment of temperatures
  • Additional reagents and equipment for paraffin embedding (Zeller, )

Alternate Protocol 1: Preparation of Frozen Sections for QFISH

  Materials
  • Slides containing lightly fixed tissue sections (see protocol 1)
  • 10 mM sodium citrate, pH 6.5 (see recipe)
  • 1% pepsin working solution (see recipe), freshly prepared
  • 25%, 50%, and 95% ethanol
  • Phosphate‐buffered saline (PBS), pH 7.2 (see recipe)
  • 10 mg/ml RNase solution (see recipe)
  • PNA/centromere probe solution (see recipe)
  • 70% formamide buffer (see recipe)
  • Tween 20 buffer (see recipe)
  • TOTO‐3 DNA stain working solution (see recipe)
  • Vectashield mounting medium (Vector Labs)
  • Clear nail polish
  • Nitrogen source
  • Coplin jars
  • Heating blocks for digital adjustment of temperatures
  • Water bath that can be adjusted to 90°C
  • HybriWell hybridization sealing system for 50 to 100 µl (Molecular Probes)
  • Coverslips

Basic Protocol 2: Basic QFISH Staining Procedure

  • Formalin‐fixed paraffin‐embedded tissue of interest
  • 70% formamide buffer with blocking reagent (optional; see recipe)

Alternate Protocol 2: Modified QFISH for Formalin‐Fixed Archival Tissue

  • Cultured cells of interest
  • Hypotonic solution (see recipe)
  • Methacarn (3 parts absolute methanol/1 part glacial acetic acid), ice cold
  • 1% paraformaldehyde (see recipe)
  • Nitrogen source
  • Tabletop centrifuge
  • Glass microscope slides
  • Diamond pen

Alternate Protocol 3: Preparation of Cultured Cells for QFISH

  • Cultured cells of interest
  • 0.5% paraformaldehyde (see recipe)
  • 3% Sea Plaque GTG low‐melting agarose (FMC) in 1× TAE buffer ( appendix 2A)
  • Tabletop centrifuge
  • Toothpicks
  • 80°C water bath

Alternate Protocol 4: Preparation of Cell Pellets Embedded in Paraffin Post‐Culture for QFISH

  • Frozen epithelial cell biopsies (e.g., from gastrointestinal tract)
  • Cyanoacrylate superglue (e.g., Krazy Glue)
  • Soaking solution (see recipe)
  • Shaking solution (see recipe)
  • Methacarn (3 parts absolute methanol: 1 part glacial acetic acid)
  • Petri dish
  • Needles for sample manipulation
  • Wooden stick
  • 15‐ml centrifuge tube
  • Heat block
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Figures

Videos

Literature Cited

Literature Cited
   Allshire, R.C., Dempster, M., and Hastie, N.D. 1989. Human telomeres contain at least three types of G‐rich repeat distrbuted non‐randomly. Nucleic Acids Res. 17:4611‐4627.
   Blackburn, E.H. 1991. Structure and function of telomeres. Nature 350:569‐572.
   Brown, W.R., MacKinnon, P.J., Villasante, A., Spurr, N., Buckle, V.J., and Dobson, M.J. 1990. Structure and polymorphism of human telomere‐associated DNA. Cell 63:119‐132.
   Dean, P.N. 1980. A simplified method of DNA distribution analysis. Cell Tissue Kinet. 13:299‐308.
   Meeker, A.K., Hicks, J.L., Platz, E.A., March, G.E., Bennett, C.J., Delannoy, M.J., and De Marzo, A.M. 2002a. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62:6405‐6409.
   Meeker, A.K., Gage, W.R., Hicks, J.L., Simon, I., Coffman, J.R., Platz, E.A., March, G.E., and De Marzo, A.M. 2002b. Telomere length assessment in human archival tissues: Combined telomere fluorescence in situ hybridization and immunostaining. Am. J. Pathol. 160:1259‐1268.
   Oexle, K. 1998. Telomere length distribution and Southern blot analysis. J. Theor. Biol. 190:369‐377.
   O'Sullivan, J.N., Bronner, M.P., Brentnall, T.A., Finley, J.C., Shen, W.T., Emerson, S., Emond, M.J., Gollahon, K.A., Moskovitz, A.H., Crispin, D.A., Potter, J.D., and Rabinovitch, P.S. 2002. Chromosomal instability in ulcerative colitis is related to telomere shortening. Nat. Genet. 32:280‐284.
   O'Sullivan, J.N., Finley, J.C., Risques, R.A., Shen, W.T., Gollahon, K.A., Moskovitz, A.H., Gryaznov, S., Harley, C.B., and Rabinovitch, P.S. 2004. Telomere length assessment in tissue sections by quantitative FISH: Image analysis algorithms. Cytometry 58A:120‐131.
   Poon, S.S., Martens, U.M., Ward, R.K., and Lansdorp, P.M. 1999. Telomere length measurements using digital fluorescence microscopy. Cytometry 36:267‐278.
   Rudolph, K.L., Millard, M., Bosenberg, M.W., and DePinho, R.A. 2001. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Nat. Genet. 28:155‐159.
   Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E., and Lansdorp, P.M. 1998. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat. Biotechnol. 16:743‐747.
   van Heek, N.T., Meeker, A.K., Kern, S.E., Yeo, C.J., Lillemoe, K.D., Cameron, J.L., Offerhaus, G.J., Hicks, J.L., Wilentz, R.E., Goggins, M.G., De Marzo, A.M., Hruban, R.H., and Maitra, A. 2002. Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia. Am. J. Pathol. 161:1541‐1547.
   Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.1.1‐14.1.8. John Wiley & Sons, Hoboken, N.J.
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