Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.)
Identifieur interne : 000D27 ( Main/Exploration ); précédent : 000D26; suivant : 000D28Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.)
Auteurs : Rudy Huis [France] ; Simon Hawkins [France] ; Godfrey Neutelings [France]Source :
- BMC Plant Biology [ 1471-2229 ] ; 2010.
Abstract
Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (
Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.
qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax
The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.
Url:
DOI: 10.1186/1471-2229-10-71
PubMed: 20403198
PubMed Central: 3095345
Affiliations:
- France
- Hauts-de-France, Nord-Pas-de-Calais
- Villeneuve d'Ascq
- Université Lille I - Sciences et technologies
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SN2, Université Lille 1, F-59655 Villeneuve d'Ascq, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Bât. SN2, Université Lille 1, F-59655 Villeneuve d'Ascq</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Villeneuve d'Ascq</settlement></placeName><orgName type="university">Université Lille I - Sciences et technologies</orgName></affiliation></author><author><name sortKey="Hawkins, Simon" sort="Hawkins, Simon" uniqKey="Hawkins S" first="Simon" last="Hawkins">Simon Hawkins</name><affiliation wicri:level="4"><nlm:aff id="I1">UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Bât. SN2, Université Lille 1, F-59655 Villeneuve d'Ascq, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Bât. SN2, Université Lille 1, F-59655 Villeneuve d'Ascq</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Villeneuve d'Ascq</settlement></placeName><orgName type="university">Université Lille I - Sciences et technologies</orgName></affiliation></author><author><name sortKey="Neutelings, Godfrey" sort="Neutelings, Godfrey" uniqKey="Neutelings G" first="Godfrey" last="Neutelings">Godfrey Neutelings</name><affiliation wicri:level="4"><nlm:aff id="I1">UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Bât. SN2, Université Lille 1, F-59655 Villeneuve d'Ascq, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Bât. SN2, Université Lille 1, F-59655 Villeneuve d'Ascq</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Villeneuve d'Ascq</settlement></placeName><orgName type="university">Université Lille I - Sciences et technologies</orgName></affiliation></author></analytic><series><title level="j">BMC Plant Biology</title><idno type="eISSN">1471-2229</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (<italic>Linum usitatissimum </italic>L).</p></sec><sec><title>Results</title><p>Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.</p><p>qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax <italic>LuMYB1 </italic>gene (homologue of <italic>AtMYB59</italic>). <italic>LuMYB1-1 </italic>and <italic>LuMYB1-2 </italic>were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.</p></sec><sec><title>Conclusions</title><p>The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zohary, D" uniqKey="Zohary D">D Zohary</name></author><author><name sortKey="Hopf, M" uniqKey="Hopf M">M Hopf</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Bodros, E" uniqKey="Bodros E">E Bodros</name></author><author><name sortKey="Pillin, I" uniqKey="Pillin I">I Pillin</name></author><author><name sortKey="Montrley, N" uniqKey="Montrley N">N Montrley</name></author><author><name sortKey="Baley, C" uniqKey="Baley C">C Baley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorshkova, Ta" uniqKey="Gorshkova T">TA Gorshkova</name></author><author><name sortKey="Wyatt, Se" uniqKey="Wyatt S">SE Wyatt</name></author><author><name 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Du</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rozen, S" uniqKey="Rozen S">S Rozen</name></author><author><name sortKey="Skaletsky, H" uniqKey="Skaletsky H">H Skaletsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zuker, M" uniqKey="Zuker M">M Zuker</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>France</li></country><region><li>Hauts-de-France</li><li>Nord-Pas-de-Calais</li></region><settlement><li>Villeneuve d'Ascq</li></settlement><orgName><li>Université Lille I - Sciences et technologies</li></orgName></list><tree><country name="France"><region name="Hauts-de-France"><name sortKey="Huis, Rudy" sort="Huis, Rudy" uniqKey="Huis R" first="Rudy" last="Huis">Rudy Huis</name></region><name sortKey="Hawkins, Simon" sort="Hawkins, Simon" uniqKey="Hawkins S" first="Simon" last="Hawkins">Simon Hawkins</name><name sortKey="Neutelings, Godfrey" sort="Neutelings, 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