Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense
Identifieur interne : 001694 ( Ncbi/Merge ); précédent : 001693; suivant : 001695Evaluation 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>
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<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>
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<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>
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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>
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