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Automated Five-Color Multiplex Co-detection of MicroRNA and Protein Expression in Fixed Tissue Specimens

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In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2148))

Abstract

microRNAs are an important class of noncoding regulatory RNAs with functional roles in development, physiology, and disease. Visualization of microRNA expression at a single-cell level has contributed to a better understanding of their biological function in animal models and their etiological contribution to human diseases. In addition, several microRNAs have been highlighted as potential biomarkers carrying diagnostic and prognostic information. Co-detection of microRNA expression with that of cell-type-specific proteins can enhance the interpretative power of expression changes during development or altered expression in pathological conditions. Here, we describe an automated fluorescence-based five-color multiplex assay for co-detection of microRNA (e.g., miR-10b, miR-21, miR-205), noncoding RNA (e.g., snRNA U6, 18S rRNA), and protein expression (e.g., cytokeratin 19, vimentin, collagen I) in paraffin-embedded formalin-fixed tissue slides on a Leica Bond Rx staining station. While this protocol uses mainly mouse tissues to demonstrate multiplex detection, it can be generally applied to single-cell expression analysis of other animal models and clinical specimens.

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Acknowledgments

We would like to thank Scot Marsh at Leica Biosystems for technical assistance and suggestions during assay development. We would like to thank Tom Wood and other members of MSU Veterinary Diagnostics Laboratory for technical assistance in tissue procurement and preparation. This work was supported, in part, by National Cancer Institute R21 CA226579 grant to LS and by Phi Beta Psi Sorority to LS.

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Authors and Affiliations

  1. Precision Health Program, Michigan State University, East Lansing, MI, USA

    Lorenzo F. Sempere, Erin Zaluzec, Elizabeth Kenyon & Anna Moore

  2. Veterinary Diagnostic Laboratory, Michigan State University, Lansing, MI, USA

    Matti Kiupel

Authors
  1. Lorenzo F. Sempere
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  2. Erin Zaluzec
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  3. Elizabeth Kenyon
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  4. Matti Kiupel
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  5. Anna Moore
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Corresponding author

Correspondence to Lorenzo F. Sempere .

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Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

  2. Cancer Research UK, University of Cambridge, Cambridge, UK

    Julia Jones

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Sempere, L.F., Zaluzec, E., Kenyon, E., Kiupel, M., Moore, A. (2020). Automated Five-Color Multiplex Co-detection of MicroRNA and Protein Expression in Fixed Tissue Specimens. In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_17

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  • DOI: https://doi.org/10.1007/978-1-0716-0623-0_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0622-3

  • Online ISBN: 978-1-0716-0623-0

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