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Fluorescence In Situ Hybridization Analysis of Formalin Fixed Paraffin Embedded Tissues, Including Tissue Microarrays

  • Brenda M. Summersgill
  • Janet M. ShipleyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Abstract

Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2–3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

Key words

Fluorescence in situ hybridization Formalin fixed paraffin embedded Tissue microarrays Genomic aberrations 

Notes

Acknowledgments

The authors would like to thank Drs. Jeremy Clark and Sian Rizzo for their helpful comments.

References

  1. 1.
    Shipley, J. and Fisher, C. (1998) Chromosome translocations in sarcomas and the analysis of paraffin-embedded material. J. Pathol. 184, 1–3.PubMedCrossRefGoogle Scholar
  2. 2.
    Summersgill, B., Clark, J. and Shipley, J. (2008) Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays. Nat. Protoc. 3, 220–234.PubMedCrossRefGoogle Scholar
  3. 3.
    Srinivasan, M., Sedmak, D. and Jewell, S. (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161, 1961–1971.PubMedCrossRefGoogle Scholar
  4. 4.
    Bramwell, N.H. and Burns, B.F. (1988) The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. Exp. Hematol. 16, 730–732.PubMedGoogle Scholar
  5. 5.
    Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 1–3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  6. 6.
    Kononen, J. Bubendorf, L., Kallioniemi, A., Bärlund, M., Schraml, P., Leighton, S., Torhorst, J., Mihatsch, M.J., Sauter, G. and Kallioniemi, O.P. (1998). Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med. 4, 844–847.PubMedCrossRefGoogle Scholar
  7. 7.
    Smedley, D., Sidhar, S., Birdsall, S., Bennett, D., Herlyn, M., Cooper, C. and Shipley, J. (2000) Characterization of chromosome 1 abnormalities in malignant melanomas. Genes Chromosomes Cancer 28, 121–125.PubMedCrossRefGoogle Scholar
  8. 8.
    Lu, Y.J., Birdsall, S., Summersgill, B., Smedley, D., Osin, P., Fisher, C. and Shipley, J. (1999) Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11.2;q11.2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma. J. Pathol. 187, 490–496.PubMedCrossRefGoogle Scholar
  9. 9.
    Birdsall, S., Osin, P., Lu, Y.J., Fisher, C. and Shipley, J. (1999) Synovial sarcoma specific translocation associated with both epithelial and spindle cell components. Int. J. Cancer 82, 605–608.PubMedCrossRefGoogle Scholar
  10. 10.
    Lambros, M.B., Simpson, P.T., Jones, C., Natrajan, R., Westbury, C., Steele, D., Savage, K., Mackay, A., Schmitt, F.C., Ashworth, A. and Reis-Filho, J.S. (2006) Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization. Lab. Invest. 86, 398–408.PubMedCrossRefGoogle Scholar
  11. 11.
    Schraml, P., Kononen, J., Bubendorf, L., Moch, H., Bissig, H., Nocito, A., Mihatsch, M.J., Kallioniemi, O.P. and Sauter, G. (1999) Tissue microarrays for gene amplification surveys in many different tumor types. Clin. Cancer Res. 5, 1966–1975.PubMedGoogle Scholar
  12. 12.
    Brown, L.A. and Huntsman, D. (2007) Fluorescent in situ hybridization on tissue microarrays: challenges and solutions. J. Mol. Histol. 38, 151–157.PubMedCrossRefGoogle Scholar
  13. 13.
    Ventura, R.A., Martin-Subero, J.I., Jones, M., McParland, J., Gesk, S., Mason, D.Y. and Siebert, R. (2006) FISH analysis for the detection of lymphoma-associated chromosomal abnormalities in routine paraffin-embedded tissue. J. Mol. Diagn. 8, 141–151.PubMedCrossRefGoogle Scholar
  14. 14.
    Bayani, J. and Squire, J.A. (2007) Application and interpretation of FISH in biomarker studies. Cancer Lett. 249, 97–109.PubMedCrossRefGoogle Scholar
  15. 15.
    Shipley, J., Crew, J., Birdsall, S., Gill, S., Clark, J., Fisher, C., Kelsey, A., Nojima, T., Sonobe, H., Cooper, C. and Gusterson, B. (1996) Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma. Am. J. Pathol. 148, 559–567.PubMedGoogle Scholar
  16. 16.
    Baschong, W., Suetterlin, R. and Laeng, R.H. (2001) Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J. Histochem. Cytochem. 49, 1565–1572.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Molecular Cytogenetics, Male Urological Cancer Research CentreInstitute of Cancer ResearchSurreyUK

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