Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos
Identifieur interne : 000260 ( Pmc/Curation ); précédent : 000259; suivant : 000261Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos
Auteurs : Karen Goossens [Belgique] ; Mario Van Poucke [Belgique] ; Ann Van Soom [Belgique] ; Jo Vandesompele [Belgique] ; Alex Van Zeveren [Belgique] ; Luc J. Peelman [Belgique]Source :
- BMC Developmental Biology [ 1471-213X ] ; 2005.
Abstract
Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published
In this study the transcription levels of 8 commonly used reference genes (
Using the geNorm application
Url:
DOI: 10.1186/1471-213X-5-27
PubMed: 16324220
PubMed Central: 1315359
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<author><name sortKey="Van Poucke, Mario" sort="Van Poucke, Mario" uniqKey="Van Poucke M" first="Mario" last="Van Poucke">Mario Van Poucke</name>
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<country xml:lang="fr">Belgique</country>
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<author><name sortKey="Van Soom, Ann" sort="Van Soom, Ann" uniqKey="Van Soom A" first="Ann" last="Van Soom">Ann Van Soom</name>
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<author><name sortKey="Goossens, Karen" sort="Goossens, Karen" uniqKey="Goossens K" first="Karen" last="Goossens">Karen Goossens</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
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<author><name sortKey="Van Poucke, Mario" sort="Van Poucke, Mario" uniqKey="Van Poucke M" first="Mario" last="Van Poucke">Mario Van Poucke</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Van Soom, Ann" sort="Van Soom, Ann" uniqKey="Van Soom A" first="Ann" last="Van Soom">Ann Van Soom</name>
<affiliation wicri:level="1"><nlm:aff id="I2">Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Vandesompele, Jo" sort="Vandesompele, Jo" uniqKey="Vandesompele J" first="Jo" last="Vandesompele">Jo Vandesompele</name>
<affiliation wicri:level="1"><nlm:aff id="I3">Center for Medical Genetics Ghent, Ghent University Hospital, Medical Research Building, De Pintelaan 185, B-9000 Ghent, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Center for Medical Genetics Ghent, Ghent University Hospital, Medical Research Building, De Pintelaan 185, B-9000 Ghent</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Van Zeveren, Alex" sort="Van Zeveren, Alex" uniqKey="Van Zeveren A" first="Alex" last="Van Zeveren">Alex Van Zeveren</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Peelman, Luc J" sort="Peelman, Luc J" uniqKey="Peelman L" first="Luc J" last="Peelman">Luc J. Peelman</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
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<series><title level="j">BMC Developmental Biology</title>
<idno type="eISSN">1471-213X</idno>
<imprint><date when="2005">2005</date>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published <italic>GAPD</italic>
, <italic>ACTB </italic>
or <italic>18S rRNA </italic>
have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used.</p>
</sec>
<sec><title>Results</title>
<p>In this study the transcription levels of 8 commonly used reference genes (<italic>ACTB</italic>
, <italic>GAPD</italic>
, <italic>Histone H2A</italic>
, <italic>TBP</italic>
, <italic>HPRT1</italic>
, <italic>SDHA</italic>
, <italic>YWHAZ </italic>
and <italic>18S rRNA</italic>
) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages.</p>
</sec>
<sec><title>Conclusion</title>
<p>Using the geNorm application <italic>YWHAZ</italic>
, <italic>GAPD </italic>
and <italic>SDHA </italic>
were found to be the most stable genes across the examined embryonic stages, while the commonly used <italic>ACTB </italic>
was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Dev Biol</journal-id>
<journal-title>BMC Developmental Biology</journal-title>
<issn pub-type="epub">1471-213X</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
<publisher-loc>London</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">16324220</article-id>
<article-id pub-id-type="pmc">1315359</article-id>
<article-id pub-id-type="publisher-id">1471-213X-5-27</article-id>
<article-id pub-id-type="doi">10.1186/1471-213X-5-27</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos</article-title>
</title-group>
<contrib-group><contrib id="A1" contrib-type="author"><name><surname>Goossens</surname>
<given-names>Karen</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Karen.Goossens@UGent.be</email>
</contrib>
<contrib id="A2" contrib-type="author"><name><surname>Van Poucke</surname>
<given-names>Mario</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Mario.VanPoucke@UGent.be</email>
</contrib>
<contrib id="A3" contrib-type="author"><name><surname>Van Soom</surname>
<given-names>Ann</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>Ann.VanSoom@UGent.be</email>
</contrib>
<contrib id="A4" contrib-type="author"><name><surname>Vandesompele</surname>
<given-names>Jo</given-names>
</name>
<xref ref-type="aff" rid="I3">3</xref>
<email>Joke.Vandesompele@UGent.be</email>
</contrib>
<contrib id="A5" contrib-type="author"><name><surname>Van Zeveren</surname>
<given-names>Alex</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Alex.VanZeveren@UGent.be</email>
</contrib>
<contrib id="A6" corresp="yes" contrib-type="author"><name><surname>Peelman</surname>
<given-names>Luc J</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Luc.Peelman@UGent.be</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium</aff>
<aff id="I2"><label>2</label>
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium</aff>
<aff id="I3"><label>3</label>
Center for Medical Genetics Ghent, Ghent University Hospital, Medical Research Building, De Pintelaan 185, B-9000 Ghent, Belgium</aff>
<pub-date pub-type="collection"><year>2005</year>
</pub-date>
<pub-date pub-type="epub"><day>3</day>
<month>12</month>
<year>2005</year>
</pub-date>
<volume>5</volume>
<fpage>27</fpage>
<lpage>27</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-213X/5/27"></ext-link>
<history><date date-type="received"><day>9</day>
<month>6</month>
<year>2005</year>
</date>
<date date-type="accepted"><day>3</day>
<month>12</month>
<year>2005</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2005 Goossens et al; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2005</copyright-year>
<copyright-holder>Goossens et al; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment>
Goossens
Karen
Karen.Goossens@UGent.be
Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos
2005 BMC Developmental Biology 5(1): 27-. (2005) 1471-213X(2005)5:1<27> urn:ISSN:1471-213X </pmc-comment>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published <italic>GAPD</italic>
, <italic>ACTB </italic>
or <italic>18S rRNA </italic>
have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used.</p>
</sec>
<sec><title>Results</title>
<p>In this study the transcription levels of 8 commonly used reference genes (<italic>ACTB</italic>
, <italic>GAPD</italic>
, <italic>Histone H2A</italic>
, <italic>TBP</italic>
, <italic>HPRT1</italic>
, <italic>SDHA</italic>
, <italic>YWHAZ </italic>
and <italic>18S rRNA</italic>
) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages.</p>
</sec>
<sec><title>Conclusion</title>
<p>Using the geNorm application <italic>YWHAZ</italic>
, <italic>GAPD </italic>
and <italic>SDHA </italic>
were found to be the most stable genes across the examined embryonic stages, while the commonly used <italic>ACTB </italic>
was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.</p>
</sec>
</abstract>
</article-meta>
</front>
</pmc>
</record>
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