Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Young, Alexander P., Jackson, Daniel J., Wyeth, Russell C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085896/
https://www.ncbi.nlm.nih.gov/pubmed/32219032
http://dx.doi.org/10.7717/peerj.8806
_version_ 1783509035471863808
author Young, Alexander P.
Jackson, Daniel J.
Wyeth, Russell C.
author_facet Young, Alexander P.
Jackson, Daniel J.
Wyeth, Russell C.
author_sort Young, Alexander P.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7085896
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher PeerJ Inc.
record_format MEDLINE/PubMed
spelling pubmed-70858962020-03-26 A technical review and guide to RNA fluorescence in situ hybridization Young, Alexander P. Jackson, Daniel J. Wyeth, Russell C. PeerJ Biochemistry 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. PeerJ Inc. 2020-03-19 /pmc/articles/PMC7085896/ /pubmed/32219032 http://dx.doi.org/10.7717/peerj.8806 Text en © 2020 Young et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biochemistry
Young, Alexander P.
Jackson, Daniel J.
Wyeth, Russell C.
A technical review and guide to RNA fluorescence in situ hybridization
title A technical review and guide to RNA fluorescence in situ hybridization
title_full A technical review and guide to RNA fluorescence in situ hybridization
title_fullStr A technical review and guide to RNA fluorescence in situ hybridization
title_full_unstemmed A technical review and guide to RNA fluorescence in situ hybridization
title_short A technical review and guide to RNA fluorescence in situ hybridization
title_sort technical review and guide to rna fluorescence in situ hybridization
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085896/
https://www.ncbi.nlm.nih.gov/pubmed/32219032
http://dx.doi.org/10.7717/peerj.8806
work_keys_str_mv AT youngalexanderp atechnicalreviewandguidetornafluorescenceinsituhybridization
AT jacksondanielj atechnicalreviewandguidetornafluorescenceinsituhybridization
AT wyethrussellc atechnicalreviewandguidetornafluorescenceinsituhybridization
AT youngalexanderp technicalreviewandguidetornafluorescenceinsituhybridization
AT jacksondanielj technicalreviewandguidetornafluorescenceinsituhybridization
AT wyethrussellc technicalreviewandguidetornafluorescenceinsituhybridization