Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR
Identifieur interne : 000773 ( Istex/Corpus ); précédent : 000772; suivant : 000774Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR
Auteurs : S. Chey ; C. Claus ; U. G. LiebertSource :
- Journal of Cellular Biochemistry [ 0730-2312 ] ; 2010-05-01.
Abstract
Reference genes are generally employed in real‐time quantitative PCR (RT‐qPCR) experiments to normalize variability between different samples. The aim of this study was to identify and validate appropriate reference genes as internal controls for RT‐qPCR experiments in rubella virus (RV)‐infected Vero and MCF‐7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the ΔΔCt calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT‐qPCR analysis of RV‐infected Vero and MCF‐7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin‐dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV‐infected cells. The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. Biochem. 110: 118–128, 2010. © 2010 Wiley‐Liss, Inc.
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DOI: 10.1002/jcb.22518
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The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. 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The aim of this study was to identify and validate appropriate reference genes as internal controls for RT‐qPCR experiments in rubella virus (RV)‐infected Vero and MCF‐7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the ΔΔCt calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT‐qPCR analysis of RV‐infected Vero and MCF‐7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin‐dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV‐infected cells. The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. 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[2007]</json:string><json:string>Eisenberg and Levanon, 2003</json:string><json:string>Watson et al., 2007</json:string><json:string>Mo et al., 2007</json:string><json:string>Takaoka et al., 2003</json:string><json:string>Rasmussen, 2001</json:string><json:string>Glare et al., 2002</json:string><json:string>Grotzinger et al., 1996</json:string><json:string>Cicinnati et al. [2008]</json:string><json:string>Leong et al. [2005]</json:string><json:string>Silver et al. [2006]</json:string><json:string>Adamo et al., 2008</json:string><json:string>Radonic et al., 2005</json:string><json:string>Xu et al., 2009</json:string><json:string>Huggett et al., 2005</json:string><json:string>Yamane et al., 2009</json:string><json:string>de Jonge et al., 2007</json:string><json:string>Ilkow et al., 2008</json:string><json:string>Vandesompele et al., 2009</json:string><json:string>Diaz et al., 1988</json:string><json:string>Takahashi et al. [2007]</json:string><json:string>Fu et al., 2009</json:string><json:string>Ilkow et al.</json:string><json:string>Beatch et al., 2005</json:string><json:string>Kasahara et al. [2005]</json:string><json:string>[2005]</json:string><json:string>Nygaard and Hovig, 2009</json:string><json:string>Mo et al.</json:string><json:string>Bernasconi et al., 2006</json:string><json:string>Yuan et al. [2007]</json:string><json:string>Skovgaard et al., 2007</json:string><json:string>Vandesompele et al., 2002</json:string><json:string>Bustin et al., 2009</json:string><json:string>Masek et al., 2005</json:string><json:string>[2004]</json:string><json:string>Vandesompele et al. 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The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin‐dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV‐infected cells. The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. Biochem. 110: 118–128, 2010. © 2010 Wiley‐Liss, Inc.</p></abstract><textClass><keywords xml:lang="en"><term xml:id="kwd1">reference genes</term><term xml:id="kwd2">normalization factors</term><term xml:id="kwd3">rubella virus</term><term xml:id="kwd4">quantitative real‐time PCR</term><term xml:id="kwd5">gene expression</term></keywords><keywords rend="articleCategory"><term>Article</term></keywords><keywords rend="tocHeading1"><term>Articles</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2019-12-20" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-DCH4H5CX-5/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>Wiley Subscription Services, Inc., A Wiley Company</publisherName><publisherLoc>Hoboken</publisherLoc></publisherInfo><doi registered="yes">10.1002/(ISSN)1097-4644</doi><issn type="print">0730-2312</issn><issn type="electronic">1097-4644</issn><idGroup><id type="product" value="JCB"></id></idGroup><titleGroup><title type="main" xml:lang="en" sort="JOURNAL OF CELLULAR BIOCHEMISTRY">Journal of Cellular Biochemistry</title><title type="short">J. 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Biochem.</title></titleGroup></publicationMeta><publicationMeta level="part" position="10"><doi origin="wiley" registered="yes">10.1002/jcb.v110:1</doi><numberingGroup><numbering type="journalVolume" number="110">110</numbering><numbering type="journalIssue">1</numbering></numberingGroup><coverDate startDate="2010-05-01">1 May 2010</coverDate></publicationMeta><publicationMeta level="unit" type="article" position="140" status="forIssue"><doi origin="wiley" registered="yes">10.1002/jcb.22518</doi><idGroup><id type="unit" value="JCB22518"></id></idGroup><countGroup><count type="pageTotal" number="11"></count></countGroup><titleGroup><title type="articleCategory">Article</title><title type="tocHeading1">Articles</title></titleGroup><copyright ownership="publisher">Copyright © 2010 Wiley‐Liss, Inc.</copyright><eventGroup><event type="manuscriptReceived" date="2009-10-14"></event><event type="manuscriptAccepted" date="2010-01-06"></event><event type="publishedOnlineEarlyUnpaginated" date="2010-03-09"></event><event type="firstOnline" date="2010-03-09"></event><event type="publishedOnlineFinalForm" date="2010-04-16"></event><event type="xmlConverted" agent="Converter:JWSART34_TO_WML3G version:2.3.15 mode:FullText source:FullText result:FullText mathml2tex" date="2010-07-21"></event><event type="xmlConverted" agent="Converter:WILEY_ML3G_TO_WILEY_ML3GV2 version:3.8.8" date="2014-01-29"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.1.7 mode:FullText,remove_FC" date="2014-10-23"></event></eventGroup><numberingGroup><numbering type="pageFirst">118</numbering><numbering type="pageLast">128</numbering></numberingGroup><correspondenceTo>Institute of Virology, University of Leipzig, Leipzig, Germany.</correspondenceTo><linkGroup><link type="toTypesetVersion" href="file:JCB.JCB22518.pdf"></link></linkGroup></publicationMeta><contentMeta><countGroup><count type="figureTotal" number="6"></count><count type="tableTotal" number="3"></count><count type="referenceTotal" number="33"></count><count type="wordTotal" number="8192"></count></countGroup><titleGroup><title type="main" xml:lang="en">Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR<link href="#fn1"></link></title><title type="short" xml:lang="en">NORMALIZATION OF RV‐INFECTED CELL LINES</title></titleGroup><creators><creator xml:id="au1" creatorRole="author" affiliationRef="#af1"><personName><givenNames>S.</givenNames><familyName>Chey</familyName></personName></creator><creator xml:id="au2" creatorRole="author" affiliationRef="#af1"><personName><givenNames>C.</givenNames><familyName>Claus</familyName></personName></creator><creator xml:id="au3" creatorRole="author" affiliationRef="#af1" corresponding="yes"><personName><givenNames>U.G.</givenNames><familyName>Liebert</familyName></personName><contactDetails><email normalForm="liebert@medizin.uni-leipzig.de">liebert@medizin.uni‐leipzig.de</email></contactDetails></creator></creators><affiliationGroup><affiliation xml:id="af1" countryCode="DE" type="organization"><unparsedAffiliation>Institute of Virology, University of Leipzig, Leipzig, Germany</unparsedAffiliation></affiliation></affiliationGroup><keywordGroup xml:lang="en" type="author"><keyword xml:id="kwd1">reference genes</keyword><keyword xml:id="kwd2">normalization factors</keyword><keyword xml:id="kwd3">rubella virus</keyword><keyword xml:id="kwd4">quantitative real‐time PCR</keyword><keyword xml:id="kwd5">gene expression</keyword></keywordGroup><fundingInfo><fundingAgency>Medical Faculty, University of Leipzig (Junior Research Grant)</fundingAgency></fundingInfo><supportingInformation><p>
Additional Supporting Information may be found in the online version of this article.
</p><supportingInfoItem><mediaResource alt="supporting information" href="urn-x:wiley:07302312:media:jcb22518:JCB_22518_sm_SupplFig1"></mediaResource><caption>Supplementary Figure 1. RNA quality assessment based on ribosomal RNA band integrity. RNA integrity of each sample of isolated total cellular RNA was analyzed by denaturing agarose electrophoresis. Shown are representative examples for mock‐ and RV‐infected Vero and MCF‐7 cells (5 µg) of total RNA were resolved on each lane along with the RiboRulerTMHigh Range RNA Ladder (Fermentas).</caption></supportingInfoItem></supportingInformation><abstractGroup><abstract type="main" xml:lang="en"><title type="main">Abstract</title><p>Reference genes are generally employed in real‐time quantitative PCR (RT‐qPCR) experiments to normalize variability between different samples. The aim of this study was to identify and validate appropriate reference genes as internal controls for RT‐qPCR experiments in rubella virus (RV)‐infected Vero and MCF‐7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the ΔΔC<sub>t</sub> calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT‐qPCR analysis of RV‐infected Vero and MCF‐7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin‐dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV‐infected cells. The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. Biochem. 110: 118–128, 2010. © 2010 Wiley‐Liss, Inc.</p></abstract></abstractGroup></contentMeta><noteGroup><note xml:id="fn1"><p>S. Chey and C. Claus contributed equally to this work.</p></note></noteGroup></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>NORMALIZATION OF RV‐INFECTED CELL LINES</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR</title></titleInfo><name type="personal"><namePart type="given">S.</namePart><namePart type="family">Chey</namePart><affiliation>Institute of Virology, University of Leipzig, Leipzig, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C.</namePart><namePart type="family">Claus</namePart><affiliation>Institute of Virology, University of Leipzig, Leipzig, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">U.G.</namePart><namePart type="family">Liebert</namePart><affiliation>Institute of Virology, University of Leipzig, Leipzig, Germany</affiliation><affiliation>E-mail: liebert@medizin.uni‐leipzig.de</affiliation><affiliation>Correspondence address: Institute of Virology, University of Leipzig, Leipzig, Germany.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</genre><originInfo><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><place><placeTerm type="text">Hoboken</placeTerm></place><dateIssued encoding="w3cdtf">2010-05-01</dateIssued><dateCaptured encoding="w3cdtf">2009-10-14</dateCaptured><dateValid encoding="w3cdtf">2010-01-06</dateValid><copyrightDate encoding="w3cdtf">2010</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><extent unit="figures">6</extent><extent unit="tables">3</extent><extent unit="references">33</extent><extent unit="words">8192</extent></physicalDescription><abstract lang="en">Reference genes are generally employed in real‐time quantitative PCR (RT‐qPCR) experiments to normalize variability between different samples. The aim of this study was to identify and validate appropriate reference genes as internal controls for RT‐qPCR experiments in rubella virus (RV)‐infected Vero and MCF‐7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the ΔΔCt calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT‐qPCR analysis of RV‐infected Vero and MCF‐7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin‐dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV‐infected cells. The effect on cellular gene transcription by RV‐infection seems to be cell line‐specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV‐infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT‐qPCR data and the host‐cell response to RV‐infection are strictly cell‐line specific. J. Cell. Biochem. 110: 118–128, 2010. © 2010 Wiley‐Liss, Inc.</abstract><note type="content">*S. Chey and C. Claus contributed equally to this work.</note><note type="funding">Medical Faculty, University of Leipzig (Junior Research Grant)</note><subject lang="en"><genre>keywords</genre><topic>reference genes</topic><topic>normalization factors</topic><topic>rubella virus</topic><topic>quantitative real‐time PCR</topic><topic>gene expression</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Cellular Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>J. Cell. Biochem.</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><note type="content"> Additional Supporting Information may be found in the online version of this article.Supporting Info Item: Supplementary Figure 1. RNA quality assessment based on ribosomal RNA band integrity. 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