Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos
Identifieur interne : 001752 ( Main/Exploration ); précédent : 001751; suivant : 001753Selection 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
Affiliations:
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Le document en format XML
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Peelman</name><affiliation wicri:level="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">16324220</idno><idno type="pmc">1315359</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1315359</idno><idno type="RBID">PMC:1315359</idno><idno type="doi">10.1186/1471-213X-5-27</idno><date 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Karen" uniqKey="Goossens K" first="Karen" last="Goossens">Karen Goossens</name><affiliation wicri:level="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></affiliation></author><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="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></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="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></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><wicri:noRegion>B-9000 Ghent</wicri:noRegion></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="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></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="4"><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><orgName type="university">Université de Gand</orgName><placeName><settlement type="city">Gand</settlement><region>Région flamande</region><region type="district" nuts="2">Province de Flandre-Orientale</region></placeName></affiliation></author></analytic><series><title level="j">BMC Developmental Biology</title><idno type="eISSN">1471-213X</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><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><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Belgique</li></country><region><li>Province de Flandre-Orientale</li><li>Région flamande</li></region><settlement><li>Gand</li></settlement><orgName><li>Université de Gand</li></orgName></list><tree><country name="Belgique"><region name="Région flamande"><name sortKey="Goossens, Karen" sort="Goossens, Karen" uniqKey="Goossens K" first="Karen" last="Goossens">Karen Goossens</name></region><name sortKey="Peelman, Luc J" sort="Peelman, Luc J" uniqKey="Peelman L" first="Luc J" last="Peelman">Luc J. Peelman</name><name sortKey="Van Poucke, Mario" sort="Van Poucke, Mario" uniqKey="Van Poucke M" first="Mario" last="Van Poucke">Mario Van Poucke</name><name sortKey="Van Soom, Ann" sort="Van Soom, Ann" uniqKey="Van Soom A" first="Ann" last="Van Soom">Ann Van Soom</name><name sortKey="Van Zeveren, Alex" sort="Van Zeveren, Alex" uniqKey="Van Zeveren A" first="Alex" last="Van Zeveren">Alex Van Zeveren</name><name sortKey="Vandesompele, Jo" sort="Vandesompele, Jo" uniqKey="Vandesompele J" first="Jo" last="Vandesompele">Jo Vandesompele</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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