Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense
Identifieur interne : 000831 ( Pmc/Checkpoint ); précédent : 000830; suivant : 000832Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense
Auteurs : Mary Mcmillan ; Lily PeregSource :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
Url:
DOI: 10.1371/journal.pone.0098162
PubMed: 24841066
PubMed Central: 4026395
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in <italic>Azospirillum brasilense</italic></title><author><name sortKey="Mcmillan, Mary" sort="Mcmillan, Mary" uniqKey="Mcmillan M" first="Mary" last="Mcmillan">Mary Mcmillan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Pereg, Lily" sort="Pereg, Lily" uniqKey="Pereg L" first="Lily" last="Pereg">Lily Pereg</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24841066</idno><idno type="pmc">4026395</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026395</idno><idno type="RBID">PMC:4026395</idno><idno type="doi">10.1371/journal.pone.0098162</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">001202</idno><idno type="wicri:Area/Pmc/Curation">001201</idno><idno type="wicri:Area/Pmc/Checkpoint">000831</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in <italic>Azospirillum brasilense</italic></title><author><name sortKey="Mcmillan, Mary" sort="Mcmillan, Mary" uniqKey="Mcmillan M" first="Mary" last="Mcmillan">Mary Mcmillan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Pereg, Lily" sort="Pereg, Lily" uniqKey="Pereg L" first="Lily" last="Pereg">Lily Pereg</name><affiliation><nlm:aff id="aff1"></nlm:aff></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><italic>Azospirillum brasilense</italic> is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions <italic>A. brasilense</italic> exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for <italic>A. brasilense</italic>. Here we evaluated the expression of ten candidate reference genes (<italic>16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA</italic>) in wild-type and mutant <italic>A. brasilense</italic> strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that <italic>gyrA</italic>, <italic>glyA</italic> and <italic>recA</italic> are the most stably expressed reference genes in <italic>A. brasilense</italic>. The results also suggested that the use of two reference genes (<italic>gyrA</italic> and <italic>glyA</italic>) is sufficient for effective normalisation of qRT-PCR data.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Steenhoudt, O" uniqKey="Steenhoudt O">O Steenhoudt</name></author><author><name sortKey="Vanderleyden" uniqKey="Vanderleyden">Vanderleyden</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Herschkovitz, Y" uniqKey="Herschkovitz Y">Y Herschkovitz</name></author><author><name sortKey="Lerner, A" uniqKey="Lerner A">A Lerner</name></author><author><name sortKey="Davidov, Y" uniqKey="Davidov Y">Y Davidov</name></author><author><name sortKey="Rothballer, M" uniqKey="Rothballer M">M Rothballer</name></author><author><name sortKey="Hartmann, A" uniqKey="Hartmann A">A Hartmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bashan, Y" uniqKey="Bashan Y">Y 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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">24841066</article-id><article-id pub-id-type="pmc">4026395</article-id><article-id pub-id-type="publisher-id">PONE-D-14-02204</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0098162</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>Biochemistry</subject><subj-group><subject>Nucleic Acids</subject><subject>RNA</subject></subj-group></subj-group><subj-group><subject>Biotechnology</subject><subj-group><subject>Applied Microbiology</subject></subj-group></subj-group><subj-group><subject>Cell Biology</subject><subj-group><subject>Molecular Cell Biology</subject></subj-group></subj-group><subj-group><subject>Genetics</subject><subj-group><subject>Gene Expression</subject></subj-group></subj-group><subj-group><subject>Microbiology</subject><subj-group><subject>Plant Microbiology</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in <italic>Azospirillum brasilense</italic></article-title><alt-title alt-title-type="running-head">Reference Genes for qRT-PCR in <italic>Azospirillum</italic></alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>McMillan</surname><given-names>Mary</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Pereg</surname><given-names>Lily</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>School of Science and Technology, University of New England, Armidale, New South Wales, Australia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Zhou</surname><given-names>Yunli</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Harvard Medical School, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>lily.pereg@une.edu.au</email></corresp><fn fn-type="conflict"><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: MM LP. Performed the experiments: MM. Analyzed the data: MM. Contributed reagents/materials/analysis tools: LP. Wrote the paper: MM LP.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>19</day><month>5</month><year>2014</year></pub-date><volume>9</volume><issue>5</issue><elocation-id>e98162</elocation-id><history><date date-type="received"><day>15</day><month>1</month><year>2014</year></date><date date-type="accepted"><day>29</day><month>4</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>McMillan, Pereg</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p><italic>Azospirillum brasilense</italic> is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions <italic>A. brasilense</italic> exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for <italic>A. brasilense</italic>. Here we evaluated the expression of ten candidate reference genes (<italic>16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA</italic>) in wild-type and mutant <italic>A. brasilense</italic> strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that <italic>gyrA</italic>, <italic>glyA</italic> and <italic>recA</italic> are the most stably expressed reference genes in <italic>A. brasilense</italic>. The results also suggested that the use of two reference genes (<italic>gyrA</italic> and <italic>glyA</italic>) is sufficient for effective normalisation of qRT-PCR data.</p></abstract><funding-group><funding-statement>Funding provided by the University of New England. 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="8"></page-count></counts></article-meta></front></pmc><affiliations><list></list><tree><noCountry><name sortKey="Mcmillan, Mary" sort="Mcmillan, Mary" uniqKey="Mcmillan M" first="Mary" last="Mcmillan">Mary Mcmillan</name><name sortKey="Pereg, Lily" sort="Pereg, Lily" uniqKey="Pereg L" first="Lily" last="Pereg">Lily Pereg</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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