Optimizing Tissue Preservation for High-Resolution Confocal Imaging of Single-Molecule RNA-FISH

Nash Redmayne, Shawn L. Chavez

    Research output: Contribution to journalArticle

    Abstract

    Over the past century, formalin-fixed, paraffin-embedded (FFPE) tissue samples have represented the standard for basic histology and immunostaining. However, FFPE has several limitations and less stringent tissue preservation methods are required for the visualization of nucleic acids at high resolution, particularly those that are expressed at low levels. Here, we describe the FFPE properties that negatively impact RNA integrity, an alternative tissue preservation technique that prevents RNA loss, and the steps necessary to optimize slide preparation for single-molecule RNA fluorescent in situ hybridization (smRNA-FISH) and imaging by confocal microscopy. This strategy retains RNA quality and eliminates formalin-induced artifacts, thereby producing high-resolution, diffraction-limited confocal images of even rare RNA transcripts in tissues. As non-coding RNAs and alternative splicing of gene isoforms continue to emerge as important regulators of human health and disease, a reliable, cost-effective approach is required to examine the expression and localization of RNA targets in patient samples.

    Original languageEnglish (US)
    Pages (from-to)e107
    JournalCurrent protocols in molecular biology
    Volume129
    Issue number1
    DOIs
    Publication statusPublished - Dec 1 2019

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    Keywords

    • alcohol fixation
    • formalin fixation
    • RNA degradation
    • single-molecule RNA-FISH
    • tissue preservation

    ASJC Scopus subject areas

    • Molecular Biology

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