Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of Spodoptera frugiperda Cells during AcMNPV Infection
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Auteurs : Tamer Z. Salem [Égypte] ; Walaa R. Allam [Égypte] ; Suzanne M. Thiem [États-Unis]Source :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection.
Url:
DOI: 10.1371/journal.pone.0108516
PubMed: 25313905
PubMed Central: 4196776
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of <italic>Spodoptera frugiperda</italic> Cells during AcMNPV Infection</title><author><name sortKey="Salem, Tamer Z" sort="Salem, Tamer Z" uniqKey="Salem T" first="Tamer Z." last="Salem">Tamer Z. Salem</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza</wicri:regionArea></affiliation></author><author><name sortKey="Allam, Walaa R" sort="Allam, Walaa R" uniqKey="Allam W" first="Walaa R." last="Allam">Walaa R. Allam</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza</wicri:regionArea></affiliation></author><author><name sortKey="Thiem, Suzanne M" sort="Thiem, Suzanne M" uniqKey="Thiem S" first="Suzanne M." last="Thiem">Suzanne M. Thiem</name><affiliation wicri:level="1"><nlm:aff id="aff3"><addr-line>Department of Entomology, Michigan State University, East Lansing, Michigan, United States of America</addr-line></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Entomology, Michigan State University, East Lansing, Michigan</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff4"><addr-line>Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America</addr-line></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25313905</idno><idno type="pmc">4196776</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196776</idno><idno type="RBID">PMC:4196776</idno><idno type="doi">10.1371/journal.pone.0108516</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000A23</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A23</idno><idno type="wicri:Area/Pmc/Curation">000A23</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000A23</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of <italic>Spodoptera frugiperda</italic> Cells during AcMNPV Infection</title><author><name sortKey="Salem, Tamer Z" sort="Salem, Tamer Z" uniqKey="Salem T" first="Tamer Z." last="Salem">Tamer Z. Salem</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza</wicri:regionArea></affiliation></author><author><name sortKey="Allam, Walaa R" sort="Allam, Walaa R" uniqKey="Allam W" first="Walaa R." last="Allam">Walaa R. Allam</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt</addr-line></nlm:aff><country xml:lang="fr">Égypte</country><wicri:regionArea>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza</wicri:regionArea></affiliation></author><author><name sortKey="Thiem, Suzanne M" sort="Thiem, Suzanne M" uniqKey="Thiem S" first="Suzanne M." last="Thiem">Suzanne M. Thiem</name><affiliation wicri:level="1"><nlm:aff id="aff3"><addr-line>Department of Entomology, Michigan State University, East Lansing, Michigan, United States of America</addr-line></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Entomology, Michigan State University, East Lansing, Michigan</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff4"><addr-line>Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America</addr-line></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan</wicri:regionArea></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. <italic>Autographa californica</italic> nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of <italic>Spodoptera frugiperda</italic> cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Thellin, O" uniqKey="Thellin O">O Thellin</name></author><author><name sortKey="Zorzi, W" uniqKey="Zorzi W">W Zorzi</name></author><author><name sortKey="Lakaye, B" uniqKey="Lakaye B">B Lakaye</name></author><author><name sortKey="De Borman, B" uniqKey="De Borman B">B De Borman</name></author><author><name sortKey="Coumans, B" uniqKey="Coumans B">B Coumans</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Radonic, A" uniqKey="Radonic A">A Radonic</name></author><author><name sortKey="Thulke, S" uniqKey="Thulke S">S Thulke</name></author><author><name sortKey="Bae, Hg" uniqKey="Bae H">HG 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25313905</article-id><article-id pub-id-type="pmc">4196776</article-id><article-id pub-id-type="publisher-id">PONE-D-14-22122</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0108516</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Microbiology</subject><subj-group><subject>Virology</subject><subj-group><subject>Virus Effects on Host Gene Expression</subject></subj-group></subj-group></subj-group><subj-group><subject>Molecular Biology</subject><subj-group><subject>Molecular Biology Techniques</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of <italic>Spodoptera frugiperda</italic> Cells during AcMNPV Infection</article-title><alt-title alt-title-type="running-head">Stable Gene Expression during Baculovirus Infection</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Salem</surname><given-names>Tamer Z.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Allam</surname><given-names>Walaa R.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Thiem</surname><given-names>Suzanne M.</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza, Egypt</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Department of Entomology, Michigan State University, East Lansing, Michigan, United States of America</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Zhang</surname><given-names>Youjun</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science, China</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>tsalem@zewailcity.edu.eg</email> (TZS); <email>smthiem@msu.edu</email> (SMT)</corresp><fn fn-type="COI-statement"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: TZS SMT. Performed the experiments: TZS. Analyzed the data: TZS AWR. Contributed reagents/materials/analysis tools: TZS AWR SMT. Wrote the paper: TZS AWR SMT.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>14</day><month>10</month><year>2014</year></pub-date><volume>9</volume><issue>10</issue><elocation-id>e108516</elocation-id><history><date date-type="received"><day>20</day><month>5</month><year>2014</year></date><date date-type="accepted"><day>23</day><month>8</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 Salem et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Salem et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. <italic>Autographa californica</italic> nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of <italic>Spodoptera frugiperda</italic> cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.</p></abstract><funding-group><funding-statement>This work was supported by the University of Science and Technology at Zewail City, Egypt to TZS and National Institutes of Health Grant 5R01GM086719 to SMT. <ext-link ext-link-type="uri" xlink:href="http://taggs.hhs.gov/readinesstool/AwardDetail.cfm?s_AwardDetail=5R01GM086719-02&STATE_CODE=26&s_RecipID=0AAEA318EF070A0FAAD29E87">http://taggs.hhs.gov/readinesstool/AwardDetail.cfm?s_AwardDetail=5R01GM086719-02&STATE_CODE=26&s_RecipID=0AAEA318EF070A0FAAD29E87</ext-link>. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="7"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. Data may be found under the following identifiers (GEO Submission (GSE60739, GPL19091, GPL19092) [NCBI tracking system #17109969]).</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. Data may be found under the following identifiers (GEO Submission (GSE60739, GPL19091, GPL19092) [NCBI tracking system #17109969]).</p></notes></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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