Identification and Evaluation of Reference Genes for Quantitative Analysis of Brazilian Pine (Araucaria angustifolia Bertol. Kuntze) Gene Expression
Identifieur interne : 001C88 ( Ncbi/Merge ); précédent : 001C87; suivant : 001C89Identification and Evaluation of Reference Genes for Quantitative Analysis of Brazilian Pine (Araucaria angustifolia Bertol. Kuntze) Gene Expression
Auteurs : Paula Elbl ; Bruno V. Navarro ; Leandro F. De Oliveira ; Juliana Almeida ; Amanda C. Mosini ; André L. W. Dos Santos ; Magdalena Rossi ; Eny I. S. FlohSource :
- PLoS ONE [ 1932-6203 ] ; 2015.
Abstract
Quantitative analysis of gene expression is a fundamental experimental approach in many fields of plant biology, but it requires the use of internal controls representing constitutively expressed genes for reliable transcript quantification. In this study, we identified fifteen putative reference genes from an
Url:
DOI: 10.1371/journal.pone.0136714
PubMed: 26313945
PubMed Central: 4552031
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Identification and Evaluation of Reference Genes for Quantitative Analysis of Brazilian Pine (<italic>Araucaria angustifolia</italic>
Bertol. Kuntze) Gene Expression</title>
<author><name sortKey="Elbl, Paula" sort="Elbl, Paula" uniqKey="Elbl P" first="Paula" last="Elbl">Paula Elbl</name>
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<author><name sortKey="Navarro, Bruno V" sort="Navarro, Bruno V" uniqKey="Navarro B" first="Bruno V." last="Navarro">Bruno V. Navarro</name>
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<author><name sortKey="Almeida, Juliana" sort="Almeida, Juliana" uniqKey="Almeida J" first="Juliana" last="Almeida">Juliana Almeida</name>
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<author><name sortKey="Dos Santos, Andre L W" sort="Dos Santos, Andre L W" uniqKey="Dos Santos A" first="André L. W." last="Dos Santos">André L. W. Dos Santos</name>
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<series><title level="j">PLoS ONE</title>
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<front><div type="abstract" xml:lang="en"><p>Quantitative analysis of gene expression is a fundamental experimental approach in many fields of plant biology, but it requires the use of internal controls representing constitutively expressed genes for reliable transcript quantification. In this study, we identified fifteen putative reference genes from an <italic>A</italic>
. <italic>angustifolia</italic>
transcriptome database. Variation in transcript levels was first evaluated <italic>in silico</italic>
by comparing read counts and then by quantitative real-time PCR (qRT-PCR), resulting in the identification of six candidate genes. The consistency of transcript abundance was also calculated applying geNorm and NormFinder software packages followed by a validation approach using four target genes. The results presented here indicate that a diverse set of samples should ideally be used in order to identify constitutively expressed genes, and that the use of any two reference genes in combination, of the six tested genes, is sufficient for effective expression normalization. Finally, in agreement with the <italic>in silico</italic>
prediction, a comprehensive analysis of the qRT-PCR data combined with validation analysis revealed that <italic>AaEIF4B-L</italic>
and <italic>AaPP2A</italic>
are the most suitable reference genes for comparative studies of <italic>A</italic>
. <italic>angustifolia</italic>
gene expression.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<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, CA USA</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">26313945</article-id>
<article-id pub-id-type="pmc">4552031</article-id>
<article-id pub-id-type="doi">10.1371/journal.pone.0136714</article-id>
<article-id pub-id-type="publisher-id">PONE-D-15-21729</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Identification and Evaluation of Reference Genes for Quantitative Analysis of Brazilian Pine (<italic>Araucaria angustifolia</italic>
Bertol. Kuntze) Gene Expression</article-title>
<alt-title alt-title-type="running-head">Brazilian Pine Reference Genes</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Elbl</surname>
<given-names>Paula</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author" equal-contrib="yes"><name><surname>Navarro</surname>
<given-names>Bruno V.</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author" equal-contrib="yes"><name><surname>de Oliveira</surname>
<given-names>Leandro F.</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Almeida</surname>
<given-names>Juliana</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Mosini</surname>
<given-names>Amanda C.</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>dos Santos</surname>
<given-names>André L. W.</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Rossi</surname>
<given-names>Magdalena</given-names>
</name>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Floh</surname>
<given-names>Eny I. S.</given-names>
</name>
<xref rid="cor001" ref-type="corresp">*</xref>
<xref ref-type="aff" rid="aff001"></xref>
</contrib>
</contrib-group>
<aff id="aff001"><addr-line>Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brasil</addr-line>
</aff>
<contrib-group><contrib contrib-type="editor"><name><surname>Chakrabarty</surname>
<given-names>Debasis</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1"><addr-line>CSIR-National Botanical Research Institute, INDIA</addr-line>
</aff>
<author-notes><fn fn-type="conflict" id="coi001"><p><bold>Competing Interests: </bold>
The authors have declared that no competing interests exist.</p>
</fn>
<fn fn-type="con" id="contrib001"><p>Conceived and designed the experiments: PE MR. Performed the experiments: PE BN LO AM. Analyzed the data: PE BN LO JA AS. Contributed reagents/materials/analysis tools: MR EF. Wrote the paper: PE BN LO EF MR.</p>
</fn>
<corresp id="cor001">* E-mail: <email>enyfloh@usp.br</email>
</corresp>
</author-notes>
<pub-date pub-type="epub"><day>27</day>
<month>8</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="collection"><year>2015</year>
</pub-date>
<volume>10</volume>
<issue>8</issue>
<elocation-id>e0136714</elocation-id>
<history><date date-type="received"><day>22</day>
<month>5</month>
<year>2015</year>
</date>
<date date-type="accepted"><day>7</day>
<month>8</month>
<year>2015</year>
</date>
</history>
<permissions><copyright-year>2015</copyright-year>
<copyright-holder>Elbl et al</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><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>
<self-uri content-type="pdf" xlink:type="simple" xlink:href="pone.0136714.pdf"></self-uri>
<abstract><p>Quantitative analysis of gene expression is a fundamental experimental approach in many fields of plant biology, but it requires the use of internal controls representing constitutively expressed genes for reliable transcript quantification. In this study, we identified fifteen putative reference genes from an <italic>A</italic>
. <italic>angustifolia</italic>
transcriptome database. Variation in transcript levels was first evaluated <italic>in silico</italic>
by comparing read counts and then by quantitative real-time PCR (qRT-PCR), resulting in the identification of six candidate genes. The consistency of transcript abundance was also calculated applying geNorm and NormFinder software packages followed by a validation approach using four target genes. The results presented here indicate that a diverse set of samples should ideally be used in order to identify constitutively expressed genes, and that the use of any two reference genes in combination, of the six tested genes, is sufficient for effective expression normalization. Finally, in agreement with the <italic>in silico</italic>
prediction, a comprehensive analysis of the qRT-PCR data combined with validation analysis revealed that <italic>AaEIF4B-L</italic>
and <italic>AaPP2A</italic>
are the most suitable reference genes for comparative studies of <italic>A</italic>
. <italic>angustifolia</italic>
gene expression.</p>
</abstract>
<funding-group><funding-statement>This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (02437/09-0), Fundação de Amparo à Pesquisa do Estado de São Paulo (2011/51659-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico(307238/2013-0).</funding-statement>
</funding-group>
<counts><fig-count count="4"></fig-count>
<table-count count="5"></table-count>
<page-count count="15"></page-count>
</counts>
<custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name>
<meta-value>All relevant data are within the paper and its Supporting Information files.</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes><title>Data Availability</title>
<p>All relevant data are within the paper and its Supporting Information files.</p>
</notes>
</front>
</pmc>
<affiliations><list></list>
<tree><noCountry><name sortKey="Almeida, Juliana" sort="Almeida, Juliana" uniqKey="Almeida J" first="Juliana" last="Almeida">Juliana Almeida</name>
<name sortKey="De Oliveira, Leandro F" sort="De Oliveira, Leandro F" uniqKey="De Oliveira L" first="Leandro F." last="De Oliveira">Leandro F. De Oliveira</name>
<name sortKey="Dos Santos, Andre L W" sort="Dos Santos, Andre L W" uniqKey="Dos Santos A" first="André L. W." last="Dos Santos">André L. W. Dos Santos</name>
<name sortKey="Elbl, Paula" sort="Elbl, Paula" uniqKey="Elbl P" first="Paula" last="Elbl">Paula Elbl</name>
<name sortKey="Floh, Eny I S" sort="Floh, Eny I S" uniqKey="Floh E" first="Eny I. S." last="Floh">Eny I. S. Floh</name>
<name sortKey="Mosini, Amanda C" sort="Mosini, Amanda C" uniqKey="Mosini A" first="Amanda C." last="Mosini">Amanda C. Mosini</name>
<name sortKey="Navarro, Bruno V" sort="Navarro, Bruno V" uniqKey="Navarro B" first="Bruno V." last="Navarro">Bruno V. Navarro</name>
<name sortKey="Rossi, Magdalena" sort="Rossi, Magdalena" uniqKey="Rossi M" first="Magdalena" last="Rossi">Magdalena Rossi</name>
</noCountry>
</tree>
</affiliations>
</record>
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