Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV
Identifieur interne : 000763 ( new/Analysis ); précédent : 000762; suivant : 000764Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV
Auteurs : Hua Yue [République populaire de Chine] ; Xiao-Wen Lei [République populaire de Chine] ; Fa-Long Yang [République populaire de Chine] ; Ming-Yi Li [République populaire de Chine] ; Cheng Tang [République populaire de Chine]Source :
- Virologica Sinica [ 1674-0769 ] ; 2010.
Abstract
Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.
Url:
DOI: 10.1007/s12250-010-3114-4
PubMed: 21221921
PubMed Central: 7090763
Affiliations:
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institution-id-type="ISNI">000000040604889X</institution-id><institution>College of Life Science and Technology,</institution><institution>Southwest University for Nationalities,</institution></institution-wrap>Chengdu, 610041 China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Chengdu</wicri:regionArea></affiliation></author><author><name sortKey="Lei, Xiao Wen" sort="Lei, Xiao Wen" uniqKey="Lei X" first="Xiao-Wen" last="Lei">Xiao-Wen Lei</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.412723.1</institution-id><institution-id institution-id-type="ISNI">000000040604889X</institution-id><institution>College of Life Science and Technology,</institution><institution>Southwest University for Nationalities,</institution></institution-wrap>Chengdu, 610041 China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Chengdu</wicri:regionArea></affiliation></author><author><name sortKey="Yang, Fa Long" sort="Yang, Fa Long" uniqKey="Yang F" first="Fa-Long" last="Yang">Fa-Long Yang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.412723.1</institution-id><institution-id institution-id-type="ISNI">000000040604889X</institution-id><institution>College of Life Science and Technology,</institution><institution>Southwest University for Nationalities,</institution></institution-wrap>Chengdu, 610041 China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Chengdu</wicri:regionArea></affiliation></author><author><name sortKey="Li, Ming Yi" sort="Li, Ming Yi" uniqKey="Li M" first="Ming-Yi" last="Li">Ming-Yi Li</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.414245.2</institution-id><institution>China Animal Health and Epidemiology Center,</institution></institution-wrap>Qingdao, 266032 China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Qingdao</wicri:regionArea></affiliation></author><author><name sortKey="Tang, Cheng" sort="Tang, Cheng" uniqKey="Tang C" first="Cheng" last="Tang">Cheng Tang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.412723.1</institution-id><institution-id institution-id-type="ISNI">000000040604889X</institution-id><institution>College of Life Science and Technology,</institution><institution>Southwest University for Nationalities,</institution></institution-wrap>Chengdu, 610041 China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Chengdu</wicri:regionArea></affiliation></author></analytic><series><title level="j">Virologica Sinica</title><idno type="ISSN">1674-0769</idno><idno type="eISSN">1995-820X</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID<sub>50</sub> of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Al Azemi, A" uniqKey="Al Azemi A">A. Al-Azemi</name></author><author><name sortKey="Bahl, J" uniqKey="Bahl J">J. Bahl</name></author><author><name sortKey="Al Zenki, S" uniqKey="Al Zenki S">S. Al-Zenki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burnside, J" uniqKey="Burnside J">J. Burnside</name></author><author><name sortKey="Ouyang, M" uniqKey="Ouyang M">M. Ouyang</name></author><author><name sortKey="Anderson, A" uniqKey="Anderson A">A. Anderson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, S A" uniqKey="Bustin S">S. A. 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Simons</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><country name="République populaire de Chine"><noRegion><name sortKey="Yue, Hua" sort="Yue, Hua" uniqKey="Yue H" first="Hua" last="Yue">Hua Yue</name></noRegion><name sortKey="Lei, Xiao Wen" sort="Lei, Xiao Wen" uniqKey="Lei X" first="Xiao-Wen" last="Lei">Xiao-Wen Lei</name><name sortKey="Li, Ming Yi" sort="Li, Ming Yi" uniqKey="Li M" first="Ming-Yi" last="Li">Ming-Yi Li</name><name sortKey="Tang, Cheng" sort="Tang, Cheng" uniqKey="Tang C" first="Cheng" last="Tang">Cheng Tang</name><name sortKey="Yang, Fa Long" sort="Yang, Fa Long" uniqKey="Yang F" first="Fa-Long" last="Yang">Fa-Long Yang</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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