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Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos
BACKGROUND: Although the transcriptome of minute quantities of cells can be profiled using nucleic acid amplification techniques, it remains difficult to distinguish between active and stored messenger RNA. Transcript storage occurs at specific stages of gametogenesis and is particularly important i...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055227/ https://www.ncbi.nlm.nih.gov/pubmed/21324132 http://dx.doi.org/10.1186/1471-213X-11-8 |
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author | Scantland, Sara Grenon, Jean-Philippe Desrochers, Marie-Hélène Sirard, Marc-André Khandjian, Edward W Robert, Claude |
author_facet | Scantland, Sara Grenon, Jean-Philippe Desrochers, Marie-Hélène Sirard, Marc-André Khandjian, Edward W Robert, Claude |
author_sort | Scantland, Sara |
collection | PubMed |
description | BACKGROUND: Although the transcriptome of minute quantities of cells can be profiled using nucleic acid amplification techniques, it remains difficult to distinguish between active and stored messenger RNA. Transcript storage occurs at specific stages of gametogenesis and is particularly important in oogenesis as stored maternal mRNA is used to sustain de novo protein synthesis during the early developmental stages until the embryonic genome gets activated. In many cases, stored mRNA can be several times more abundant than mRNA ready for translation. In order to identify active mRNA in bovine oocytes, we sought to develop a method of isolating very small amounts of polyribosome mRNA. RESULTS: The proposed method is based on mixing the extracted oocyte cytoplasm with a preparation of polyribosomes obtained from a non-homologous source (Drosophila) and using sucrose density gradient ultracentrifugation to separate the polyribosomes. It involves cross-linking the non-homologous polyribosomes and neutralizing the cross-linking agent. Using this method, we show that certain stages of oocyte maturation coincide with changes in the abundance of polyribosomal mRNA but not total RNA or poly(A). We also show that the abundance of selected sequences matched changes in the corresponding protein levels. CONCLUSIONS: We report here the successful use of a method to profile mRNA present in the polyribosomal fraction obtained from as little as 75 mammalian oocytes. Polyribosomal mRNA fractionation thus provides a new tool for studying gametogenesis and early development with better representation of the underlying physiological status. |
format | Text |
id | pubmed-3055227 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30552272011-03-12 Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos Scantland, Sara Grenon, Jean-Philippe Desrochers, Marie-Hélène Sirard, Marc-André Khandjian, Edward W Robert, Claude BMC Dev Biol Methodology Article BACKGROUND: Although the transcriptome of minute quantities of cells can be profiled using nucleic acid amplification techniques, it remains difficult to distinguish between active and stored messenger RNA. Transcript storage occurs at specific stages of gametogenesis and is particularly important in oogenesis as stored maternal mRNA is used to sustain de novo protein synthesis during the early developmental stages until the embryonic genome gets activated. In many cases, stored mRNA can be several times more abundant than mRNA ready for translation. In order to identify active mRNA in bovine oocytes, we sought to develop a method of isolating very small amounts of polyribosome mRNA. RESULTS: The proposed method is based on mixing the extracted oocyte cytoplasm with a preparation of polyribosomes obtained from a non-homologous source (Drosophila) and using sucrose density gradient ultracentrifugation to separate the polyribosomes. It involves cross-linking the non-homologous polyribosomes and neutralizing the cross-linking agent. Using this method, we show that certain stages of oocyte maturation coincide with changes in the abundance of polyribosomal mRNA but not total RNA or poly(A). We also show that the abundance of selected sequences matched changes in the corresponding protein levels. CONCLUSIONS: We report here the successful use of a method to profile mRNA present in the polyribosomal fraction obtained from as little as 75 mammalian oocytes. Polyribosomal mRNA fractionation thus provides a new tool for studying gametogenesis and early development with better representation of the underlying physiological status. BioMed Central 2011-02-15 /pmc/articles/PMC3055227/ /pubmed/21324132 http://dx.doi.org/10.1186/1471-213X-11-8 Text en Copyright ©2011 Scantland et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Scantland, Sara Grenon, Jean-Philippe Desrochers, Marie-Hélène Sirard, Marc-André Khandjian, Edward W Robert, Claude Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title | Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title_full | Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title_fullStr | Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title_full_unstemmed | Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title_short | Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos |
title_sort | method to isolate polyribosomal mrna from scarce samples such as mammalian oocytes and early embryos |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055227/ https://www.ncbi.nlm.nih.gov/pubmed/21324132 http://dx.doi.org/10.1186/1471-213X-11-8 |
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