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High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution...

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Autores principales: Takei, Natsumi, Nakamura, Takuma, Kawamura, Shohei, Takada, Yuki, Satoh, Yui, Kimura, Atsushi P., Kotani, Tomoya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831722/
https://www.ncbi.nlm.nih.gov/pubmed/29507535
http://dx.doi.org/10.1186/s12575-018-0071-z
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author Takei, Natsumi
Nakamura, Takuma
Kawamura, Shohei
Takada, Yuki
Satoh, Yui
Kimura, Atsushi P.
Kotani, Tomoya
author_facet Takei, Natsumi
Nakamura, Takuma
Kawamura, Shohei
Takada, Yuki
Satoh, Yui
Kimura, Atsushi P.
Kotani, Tomoya
author_sort Takei, Natsumi
collection PubMed
description BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12575-018-0071-z) contains supplementary material, which is available to authorized users.
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spelling pubmed-58317222018-03-05 High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes Takei, Natsumi Nakamura, Takuma Kawamura, Shohei Takada, Yuki Satoh, Yui Kimura, Atsushi P. Kotani, Tomoya Biol Proced Online Methodology BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12575-018-0071-z) contains supplementary material, which is available to authorized users. BioMed Central 2018-03-01 /pmc/articles/PMC5831722/ /pubmed/29507535 http://dx.doi.org/10.1186/s12575-018-0071-z Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Takei, Natsumi
Nakamura, Takuma
Kawamura, Shohei
Takada, Yuki
Satoh, Yui
Kimura, Atsushi P.
Kotani, Tomoya
High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title_full High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title_fullStr High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title_full_unstemmed High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title_short High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes
title_sort high-sensitivity and high-resolution in situ hybridization of coding and long non-coding rnas in vertebrate ovaries and testes
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831722/
https://www.ncbi.nlm.nih.gov/pubmed/29507535
http://dx.doi.org/10.1186/s12575-018-0071-z
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