Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization
Identifieur interne : 000677 ( Pmc/Corpus ); précédent : 000676; suivant : 000678Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization
Auteurs : Lee-Kuo Kang ; Feng-Hsiu Tsui ; Jeng ChangSource :
- Applied and Environmental Microbiology [ 0099-2240 ] ; 2012.
Abstract
To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes,
Url:
DOI: 10.1128/AEM.00935-12
PubMed: 22706063
PubMed Central: 3416636
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PMC:3416636Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization</title>
<author><name sortKey="Kang, Lee Kuo" sort="Kang, Lee Kuo" uniqKey="Kang L" first="Lee-Kuo" last="Kang">Lee-Kuo Kang</name>
<affiliation><nlm:aff id="aff1">Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tsui, Feng Hsiu" sort="Tsui, Feng Hsiu" uniqKey="Tsui F" first="Feng-Hsiu" last="Tsui">Feng-Hsiu Tsui</name>
<affiliation><nlm:aff id="aff2">Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chang, Jeng" sort="Chang, Jeng" uniqKey="Chang J" first="Jeng" last="Chang">Jeng Chang</name>
<affiliation><nlm:aff id="aff1">Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3">Center for Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization</title>
<author><name sortKey="Kang, Lee Kuo" sort="Kang, Lee Kuo" uniqKey="Kang L" first="Lee-Kuo" last="Kang">Lee-Kuo Kang</name>
<affiliation><nlm:aff id="aff1">Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tsui, Feng Hsiu" sort="Tsui, Feng Hsiu" uniqKey="Tsui F" first="Feng-Hsiu" last="Tsui">Feng-Hsiu Tsui</name>
<affiliation><nlm:aff id="aff2">Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chang, Jeng" sort="Chang, Jeng" uniqKey="Chang J" first="Jeng" last="Chang">Jeng Chang</name>
<affiliation><nlm:aff id="aff1">Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3">Center for Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Applied and Environmental Microbiology</title>
<idno type="ISSN">0099-2240</idno>
<idno type="eISSN">1098-5336</idno>
<imprint><date when="2012">2012</date>
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<front><div type="abstract" xml:lang="en"><p>To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, <italic>TBP</italic>
(encoding the TATA box-binding protein) and <italic>EFL</italic>
(encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of <italic>TBP</italic>
and <italic>EFL</italic>
were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in <named-content content-type="genus-species">Skeletonema costatum</named-content>
and <named-content content-type="genus-species">Chaetoceros affinis</named-content>
, and <italic>TBP</italic>
expression was more stable than that of <italic>EFL</italic>
. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 <italic>EFL</italic>
and 29 <italic>TBP</italic>
homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, <italic>EFL</italic>
and <italic>TBP</italic>
primer sets were designed for <named-content content-type="genus-species">Chaetoceros</named-content>
and <named-content content-type="genus-species">Skeletonema</named-content>
groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the <italic>EFL</italic>
primer sets performed better. To demonstrate the applicability of <italic>EFL</italic>
primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in <italic>FcpB</italic>
expression and confirmed <italic>EFL</italic>
as a good reference gene.</p>
</div>
</front>
</TEI>
<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Appl Environ Microbiol</journal-id>
<journal-id journal-id-type="iso-abbrev">Appl. Environ. Microbiol</journal-id>
<journal-id journal-id-type="hwp">aem</journal-id>
<journal-id journal-id-type="pmc">aem</journal-id>
<journal-id journal-id-type="publisher-id">AEM</journal-id>
<journal-title-group><journal-title>Applied and Environmental Microbiology</journal-title>
</journal-title-group>
<issn pub-type="ppub">0099-2240</issn>
<issn pub-type="epub">1098-5336</issn>
<publisher><publisher-name>American Society for Microbiology</publisher-name>
<publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">22706063</article-id>
<article-id pub-id-type="pmc">3416636</article-id>
<article-id pub-id-type="publisher-id">00935-12</article-id>
<article-id pub-id-type="doi">10.1128/AEM.00935-12</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Methods</subject>
</subj-group>
</article-categories>
<title-group><article-title>Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Kang</surname>
<given-names>Lee-Kuo</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tsui</surname>
<given-names>Feng-Hsiu</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Chang</surname>
<given-names>Jeng</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
<xref ref-type="aff" rid="aff3"><sup>c</sup>
</xref>
</contrib>
<aff id="aff1"><label>a</label>
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</aff>
<aff id="aff2"><label>b</label>
Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</aff>
<aff id="aff3"><label>c</label>
Center for Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China</aff>
</contrib-group>
<author-notes><corresp>Address correspondence to Jeng Chang, <email>jengchang@mail.ntou.edu.tw</email>
.</corresp>
</author-notes>
<pub-date pub-type="ppub"><month>9</month>
<year>2012</year>
</pub-date>
<volume>78</volume>
<issue>17</issue>
<fpage>6051</fpage>
<lpage>6058</lpage>
<history><date date-type="received"><day>22</day>
<month>3</month>
<year>2012</year>
</date>
<date date-type="accepted"><day>12</day>
<month>6</month>
<year>2012</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2012, American Society for Microbiology. All Rights Reserved.</copyright-statement>
<copyright-year>2012</copyright-year>
<copyright-holder>American Society for Microbiology</copyright-holder>
</permissions>
<self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zam01712006051.pdf"></self-uri>
<abstract><p>To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, <italic>TBP</italic>
(encoding the TATA box-binding protein) and <italic>EFL</italic>
(encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of <italic>TBP</italic>
and <italic>EFL</italic>
were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in <named-content content-type="genus-species">Skeletonema costatum</named-content>
and <named-content content-type="genus-species">Chaetoceros affinis</named-content>
, and <italic>TBP</italic>
expression was more stable than that of <italic>EFL</italic>
. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 <italic>EFL</italic>
and 29 <italic>TBP</italic>
homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, <italic>EFL</italic>
and <italic>TBP</italic>
primer sets were designed for <named-content content-type="genus-species">Chaetoceros</named-content>
and <named-content content-type="genus-species">Skeletonema</named-content>
groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the <italic>EFL</italic>
primer sets performed better. To demonstrate the applicability of <italic>EFL</italic>
primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in <italic>FcpB</italic>
expression and confirmed <italic>EFL</italic>
as a good reference gene.</p>
</abstract>
</article-meta>
</front>
</pmc>
</record>
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