Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.
Identifieur interne : 001B88 ( Main/Exploration ); précédent : 001B87; suivant : 001B89Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.
Auteurs : Maren Olbrich [Allemagne] ; Elke Gerstner ; Gerhard Welzl ; Frank Fleischmann ; Wolfgang Osswald ; Günther Bahnweg ; Dieter ErnstSource :
- Zeitschrift fur Naturforschung. C, Journal of biosciences [ 0939-5075 ]
Descripteurs français
- KwdFr :
- MESH :
- génétique : ARN messager, Fagus.
- physiologie : Fagus.
- Amorces ADN, Analyse de variance, Gènes de plante, RT-PCR, Séquence nucléotidique.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : RNA, Messenger.
- chemical : DNA Primers.
- genetics : Fagus.
- physiology : Fagus.
- Analysis of Variance, Base Sequence, Genes, Plant, Reverse Transcriptase Polymerase Chain Reaction.
Abstract
Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.
DOI: 10.1515/znc-2008-7-819
PubMed: 18811005
Affiliations:
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C, Journal of biosciences</title><idno type="ISSN">0939-5075</idno></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Analysis of Variance (MeSH)</term><term>Base Sequence (MeSH)</term><term>DNA Primers (MeSH)</term><term>Fagus (genetics)</term><term>Fagus (physiology)</term><term>Genes, Plant (MeSH)</term><term>RNA, Messenger (genetics)</term><term>Reverse Transcriptase Polymerase Chain Reaction (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (génétique)</term><term>Amorces ADN (MeSH)</term><term>Analyse de variance (MeSH)</term><term>Fagus (génétique)</term><term>Fagus (physiologie)</term><term>Gènes de plante (MeSH)</term><term>RT-PCR (MeSH)</term><term>Séquence nucléotidique (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>DNA Primers</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Fagus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN messager</term><term>Fagus</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Fagus</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>Fagus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Analysis of Variance</term><term>Base Sequence</term><term>Genes, Plant</term><term>Reverse Transcriptase Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Amorces ADN</term><term>Analyse de variance</term><term>Gènes de plante</term><term>RT-PCR</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">18811005</PMID><DateCompleted><Year>2008</Year><Month>10</Month><Day>27</Day></DateCompleted><DateRevised><Year>2019</Year><Month>10</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0939-5075</ISSN><JournalIssue CitedMedium="Print"><Volume>63</Volume><Issue>7-8</Issue><PubDate><MedlineDate>2008 Jul-Aug</MedlineDate></PubDate></JournalIssue><Title>Zeitschrift fur Naturforschung. C, Journal of biosciences</Title><ISOAbbreviation>Z Naturforsch C J Biosci</ISOAbbreviation></Journal><ArticleTitle>Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.</ArticleTitle><Pagination><MedlinePgn>574-82</MedlinePgn></Pagination><Abstract><AbstractText>Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Olbrich</LastName><ForeName>Maren</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gerstner</LastName><ForeName>Elke</ForeName><Initials>E</Initials></Author><Author ValidYN="Y"><LastName>Welzl</LastName><ForeName>Gerhard</ForeName><Initials>G</Initials></Author><Author ValidYN="Y"><LastName>Fleischmann</LastName><ForeName>Frank</ForeName><Initials>F</Initials></Author><Author ValidYN="Y"><LastName>Osswald</LastName><ForeName>Wolfgang</ForeName><Initials>W</Initials></Author><Author ValidYN="Y"><LastName>Bahnweg</LastName><ForeName>Günther</ForeName><Initials>G</Initials></Author><Author ValidYN="Y"><LastName>Ernst</LastName><ForeName>Dieter</ForeName><Initials>D</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>Germany</Country><MedlineTA>Z Naturforsch C J Biosci</MedlineTA><NlmUniqueID>8912155</NlmUniqueID><ISSNLinking>0341-0382</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D012333">RNA, Messenger</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000704" MajorTopicYN="N">Analysis of Variance</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017931" MajorTopicYN="N">DNA Primers</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D029964" MajorTopicYN="N">Fagus</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000502" MajorTopicYN="N">physiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017343" MajorTopicYN="Y">Genes, Plant</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D012333" MajorTopicYN="N">RNA, Messenger</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020133" MajorTopicYN="N">Reverse Transcriptase Polymerase Chain Reaction</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2008</Year><Month>9</Month><Day>25</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2008</Year><Month>10</Month><Day>28</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2008</Year><Month>9</Month><Day>25</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">18811005</ArticleId><ArticleId IdType="doi">10.1515/znc-2008-7-819</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Allemagne</li></country></list><tree><noCountry><name sortKey="Bahnweg, Gunther" sort="Bahnweg, Gunther" uniqKey="Bahnweg G" first="Günther" last="Bahnweg">Günther Bahnweg</name><name sortKey="Ernst, Dieter" sort="Ernst, Dieter" uniqKey="Ernst D" first="Dieter" last="Ernst">Dieter Ernst</name><name sortKey="Fleischmann, Frank" sort="Fleischmann, Frank" uniqKey="Fleischmann F" first="Frank" last="Fleischmann">Frank Fleischmann</name><name sortKey="Gerstner, Elke" sort="Gerstner, Elke" uniqKey="Gerstner E" first="Elke" last="Gerstner">Elke Gerstner</name><name sortKey="Osswald, Wolfgang" sort="Osswald, Wolfgang" uniqKey="Osswald W" first="Wolfgang" last="Osswald">Wolfgang Osswald</name><name sortKey="Welzl, Gerhard" sort="Welzl, Gerhard" uniqKey="Welzl G" first="Gerhard" last="Welzl">Gerhard Welzl</name></noCountry><country name="Allemagne"><noRegion><name sortKey="Olbrich, Maren" sort="Olbrich, Maren" uniqKey="Olbrich M" first="Maren" last="Olbrich">Maren Olbrich</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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