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Quantitative Multiplexed Quantum Dot Based In Situ Hybridization in Formalin-Fixed Paraffin-Embedded Tissue

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In Situ Hybridization Methods

Part of the book series: Neuromethods ((NM,volume 99))

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Abstract

Gene expression profiling using microarray analysis has expanded our understanding of human cancer and identified gene signatures that can be useful in predicting outcome. However, relatively little of this knowledge has been translated into clinically effective diagnostic tools since microarrays usually require high-quality fresh-frozen samples. In this chapter, a new methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot-labelled oligonucleotide probes (QD-ISH) and spectral imaging data analysis is described. Initially, optimization using a poly d(T) oligonucleotide probe in routinely processed, formalin-fixed and paraffin-embedded (FFPE) bone marrow biopsies is described. Protocols are then detailed for single and multiplex hybridization FFPE samples, illustrated by application to samples from patients with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging is used for post-hybridization image analysis, enabling separation of spatially co-localized signals. Details of application to multiplex ISH in routinely processed archival bone marrow biopsies are the described, specifically in tissue-microarrays to enable high throughput analysis and use of a standard for signal quantitation is detailed. The method enables use of standardized, quantitative multiplex QD-ISH for identification of prognostic markers in FFPE samples, and as such is generally applicable in translational research.

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Author information

Authors and Affiliations

  1. Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK

    Eleni Tholouli

  2. Centre for Regenerative Medicine, Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK

    Judith A. Hoyland

  3. Institute of Cancer Sciences, Faculty of Medical and Human Sciences, The University of Manchester, Stopford, Building, Oxford Road, Manchester, M13 9PT, UK

    Richard J. Byers

  4. Department of Histopathology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK

    Richard J. Byers

  5. Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK

    Richard J. Byers

Authors
  1. Eleni Tholouli
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  2. Judith A. Hoyland
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  3. Richard J. Byers
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Corresponding author

Correspondence to Richard J. Byers .

Editor information

Editors and Affiliations

  1. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

    Giselbert Hauptmann

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Tholouli, E., Hoyland, J.A., Byers, R.J. (2015). Quantitative Multiplexed Quantum Dot Based In Situ Hybridization in Formalin-Fixed Paraffin-Embedded Tissue. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_23

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  • DOI: https://doi.org/10.1007/978-1-4939-2303-8_23

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2302-1

  • Online ISBN: 978-1-4939-2303-8

  • eBook Packages: Springer Protocols

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