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
    StatePublished - Dec 1 2019

    Fingerprint

    Tissue Preservation
    RNA
    Formaldehyde
    Paraffin
    RNA Splicing
    Untranslated RNA
    Alternative Splicing
    Fluorescence In Situ Hybridization
    Confocal Microscopy
    Artifacts
    Nucleic Acids
    Single Molecule Imaging
    Histology
    Protein Isoforms
    Costs and Cost Analysis
    Health
    Genes

    Keywords

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

    ASJC Scopus subject areas

    • Molecular Biology

    Cite this

    Optimizing Tissue Preservation for High-Resolution Confocal Imaging of Single-Molecule RNA-FISH. / Redmayne, Nash; Chavez, Shawn L.

    In: Current protocols in molecular biology, Vol. 129, No. 1, 01.12.2019, p. e107.

    Research output: Contribution to journalArticle

    @article{75b9878e15bf4beeb25e7aba213f075e,
    title = "Optimizing Tissue Preservation for High-Resolution Confocal Imaging of Single-Molecule RNA-FISH",
    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.",
    keywords = "alcohol fixation, formalin fixation, RNA degradation, single-molecule RNA-FISH, tissue preservation",
    author = "Nash Redmayne and Chavez, {Shawn L.}",
    year = "2019",
    month = "12",
    day = "1",
    doi = "10.1002/cpmb.107",
    language = "English (US)",
    volume = "129",
    pages = "e107",
    journal = "Current Protocols in Molecular Biology",
    issn = "1934-3639",
    publisher = "John Wiley and Sons Inc.",
    number = "1",

    }

    TY - JOUR

    T1 - Optimizing Tissue Preservation for High-Resolution Confocal Imaging of Single-Molecule RNA-FISH

    AU - Redmayne, Nash

    AU - Chavez, Shawn L.

    PY - 2019/12/1

    Y1 - 2019/12/1

    N2 - 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.

    AB - 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.

    KW - alcohol fixation

    KW - formalin fixation

    KW - RNA degradation

    KW - single-molecule RNA-FISH

    KW - tissue preservation

    UR - http://www.scopus.com/inward/record.url?scp=85075527276&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=85075527276&partnerID=8YFLogxK

    U2 - 10.1002/cpmb.107

    DO - 10.1002/cpmb.107

    M3 - Article

    C2 - 31763781

    AN - SCOPUS:85075527276

    VL - 129

    SP - e107

    JO - Current Protocols in Molecular Biology

    JF - Current Protocols in Molecular Biology

    SN - 1934-3639

    IS - 1

    ER -