Model system for optimising mRNA non-isotopic in situ hybridisation: riboprobe detection of lysozyme mRNA in archival gut biopsy specimens

J. C. Martinez-Montero, C. S. Herrington, J. Stickland, H. Sawyer, M. Evans, D. M. Flannery, J. O. McGee

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    Abstract

    The aim of this study was to optimise conditions for mRNA detection by nonisotopic in situ hybridisation (NISH) using biotinylated and digoxigenin labelled riboprobes. Because lysozyme gene transcripts are present at high concentrations in Paneth and other alimentary cells, archival gut biopsy specimens were chosen as a model system for these experiments. Most of the variables in NISH, from unmasking of mRNA, to its ultimate detection by peroxidase or alkaline phosphatase based detection systems, were examined in detail. The most important findings were that simultaneous heating of tissue targets and riboprobes at 95-degrees-C for 15 minutes before hybridisation at 50-degrees-C for two hours gave the most intense signal for lysozyme mRNA in Paneth cells, Brunner's glands, and lamina propria macrophages; digoxigenin labelled riboprobes gave a higher signal to noise ratio than their biotinylated counterparts, and probes 600 base pairs long were superior to shorter probes. It is concluded that the mRNA NISH method may be generally useful for detecting gene transcription in archival clinical biopsy specimens.

    Original languageEnglish
    Pages (from-to)835-839
    Number of pages5
    JournalJournal of Clinical Pathology
    Volume44
    Issue number10
    DOIs
    Publication statusPublished - 1991

    Cite this

    Martinez-Montero, J. C., Herrington, C. S., Stickland, J., Sawyer, H., Evans, M., Flannery, D. M., & McGee, J. O. (1991). Model system for optimising mRNA non-isotopic in situ hybridisation: riboprobe detection of lysozyme mRNA in archival gut biopsy specimens. Journal of Clinical Pathology, 44(10), 835-839. https://doi.org/10.1136/jcp.44.10.835