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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

¹St Vincent's University Hospital, Elm Park, Dublin, Ireland
²University 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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Unit Introduction
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, 1989)

Basic Protocol 2: Basic QFISH Staining Procedure

Materials

Slides containing lightly fixed tissue sections (see Basic 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

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

Additional Materials (also see Basic Protocols 1 and 2)

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

Alternate Protocol 3: Preparation of Cultured Cells for QFISH

Additional Materials (also see Basic Protocols 1 and 2)

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 4: Preparation of Cell Pellets Embedded in Paraffin Post-Culture for QFISH

Additional Materials (also see Basic Protocols 1 and 2)

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 5: Preparation of Epithelial Cells Isolated from Tissue Biopsies for QFISH

Additional Materials (also see Basic Protocols 1 and 2)

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

Figure 12.6.1

(A) FISH analysis of a peripheral blood metaphase spread. Telomeres are labeled with a FITC-conjugated PNA probe (green), centromeres with a TAMRA probe (red), and DNA with a TOTO-3 DNA dye (false-colored blue). Prometaphase and interphase cells are also shown in this image. (B) Confocal QFISH image of a normal colon biopsy. (C) Biopsy of a patient with ulcerative colitis. (D) A large colon adenoma. Telomere and centromere staining intensities are equal for epithelial and stroma cells in panel B. Reduced telomere (green) fluorescence is evident in the epithelial cells of the ulcerative colitis and large-colon adenoma cases (C and D) compared to the stromal-cell telomere staining in the same section. The average intensity of the centromere and DNA staining remained unchanged between epithelial and stroma cells. The centromere and DNA probes therefore serve as internal controls for changes in telomere brightness that might have resulted from reduced accessibility to the telomere probe.

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Figure 12.6.2

Illustration of the three image-analysis algorithms described in the text: (A) background-corrected fluorescence; (B) spot-finding method; (C) background-curve subtraction.

View Image

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.