A technical review and guide to RNA fluorescence in situ hybridization

RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is...

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Bibliographic Details
Published in:	PeerJ (San Francisco, CA) CA), 2020-03, Vol.8, p.e8806-e8806, Article e8806
Main Authors:	Young, Alexander P, Jackson, Daniel J, Wyeth, Russell C
Format:	Article
Language:	eng
Subjects:	Animal behavior Automation Biochemistry Biophysics Cell Biology Deoxyribonucleic acid Divergence DNA FISH Fluorescence Fluorescence in situ hybridization Gene expression Genes Genetics Hybridization Immunoglobulins Messenger RNA Methods Mitochondrial DNA Molecular Biology mRNA mRNA expression Oligonucleotide probe Protocol Protocol development Reagents Reviews Riboprobe RNA Technology Time Visualization Whole mount
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Summary:	RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols.
ISSN:	2167-8359 2167-8359