Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV
Identifieur interne : 000353 ( Pmc/Corpus ); précédent : 000352; suivant : 000354Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV
Auteurs : Hua Yue ; Xiao-Wen Lei ; Fa-Long Yang ; Ming-Yi Li ; Cheng TangSource :
- 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
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV</title><author><name sortKey="Yue, Hua" sort="Yue, Hua" uniqKey="Yue H" first="Hua" last="Yue">Hua Yue</name><affiliation><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></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><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></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><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></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><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></affiliation></author><author><name sortKey="Tang, Cheng" sort="Tang, Cheng" uniqKey="Tang C" first="Cheng" last="Tang">Cheng Tang</name><affiliation><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></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21221921</idno><idno type="pmc">7090763</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090763</idno><idno type="RBID">PMC:7090763</idno><idno type="doi">10.1007/s12250-010-3114-4</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000353</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000353</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV</title><author><name sortKey="Yue, Hua" sort="Yue, Hua" uniqKey="Yue H" first="Hua" last="Yue">Hua Yue</name><affiliation><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></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><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></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><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></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><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></affiliation></author><author><name sortKey="Tang, Cheng" sort="Tang, Cheng" uniqKey="Tang C" first="Cheng" last="Tang">Cheng Tang</name><affiliation><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></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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<front><journal-meta><journal-id journal-id-type="nlm-ta">Virol Sin</journal-id><journal-id journal-id-type="iso-abbrev">Virol Sin</journal-id><journal-title-group><journal-title>Virologica Sinica</journal-title></journal-title-group><issn pub-type="ppub">1674-0769</issn><issn pub-type="epub">1995-820X</issn><publisher><publisher-name>SP Wuhan Institute of Virology, CAS</publisher-name><publisher-loc>Heidelberg</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21221921</article-id><article-id pub-id-type="pmc">7090763</article-id><article-id pub-id-type="publisher-id">3114</article-id><article-id pub-id-type="doi">10.1007/s12250-010-3114-4</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Yue</surname><given-names>Hua</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lei</surname><given-names>Xiao-wen</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Fa-long</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Ming-Yi</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Tang</surname><given-names>Cheng</given-names></name><address><phone>+86-28-85528276</phone><fax>+86-28-85522855</fax><email>tangcheng101@yahoo.com.cn</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label><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</aff><aff id="Aff2"><label>2</label><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</aff></contrib-group><pub-date pub-type="epub"><day>21</day><month>12</month><year>2010</year></pub-date><pub-date pub-type="collection"><month>12</month><year>2010</year></pub-date><volume>25</volume><issue>6</issue><fpage>425</fpage><lpage>431</lpage><history><date date-type="received"><day>8</day><month>11</month><year>2009</year></date><date date-type="accepted"><day>28</day><month>7</month><year>2010</year></date></history><permissions><copyright-statement>© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2010</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><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></abstract><kwd-group xml:lang="en"><title>Key words</title><kwd>Reference gene</kwd><kwd>Chicken embryo fibroblast</kwd><kwd>H5N1 avian influenza virus (AIV)</kwd><kwd>Real-time PCR (RT-PCR)</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2010</meta-value></custom-meta></custom-meta-group></article-meta></front><back><fn-group><fn><p>Foundation item: National “11th Five-year Plan” Scientific and Technical Supporting Programs (2006BAD06A11).</p></fn></fn-group><ref-list id="Bib1"><title>References</title><ref id="CR1"><label>1.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Al-Azemi</surname><given-names>A.</given-names></name><name><surname>Bahl</surname><given-names>J.</given-names></name><name><surname>Al-Zenki</surname><given-names>S.</given-names></name><etal></etal></person-group><article-title>Avian influenza A virus (H5N1) outbreaks, Kuwait, 2007</article-title><source>Emerg Infect Dis</source><year>2008</year><volume>14</volume><fpage>958</fpage><lpage>961</lpage><pub-id pub-id-type="doi">10.3201/eid1406.080056</pub-id><pub-id pub-id-type="pmid">18507914</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Burnside</surname><given-names>J.</given-names></name><name><surname>Ouyang</surname><given-names>M.</given-names></name><name><surname>Anderson</surname><given-names>A.</given-names></name><etal></etal></person-group><article-title>Deep sequencing of chicken microRNAs</article-title><source>BMC Genomics</source><year>2008</year><volume>22</volume><fpage>185</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-9-185</pub-id></element-citation></ref><ref id="CR3"><label>3.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bustin</surname><given-names>S. 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