Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar.
Identifieur interne : 003269 ( Main/Exploration ); précédent : 003268; suivant : 003270Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar.
Auteurs : Nicole Regier [Suisse] ; Beat FreySource :
- BMC molecular biology [ 1471-2199 ] ; 2010.
Descripteurs français
- KwdFr :
- Ascorbate peroxidases (MeSH), Eau (MeSH), Expression des gènes (MeSH), Normes de référence (MeSH), Peroxidases (génétique), Peroxidases (métabolisme), Populus (enzymologie), Populus (génétique), Protéines végétales (génétique), Protéines végétales (métabolisme), RT-PCR (méthodes), RT-PCR (normes), Superoxide dismutase (génétique), Superoxide dismutase (métabolisme), Sécheresses (MeSH).
- MESH :
- enzymologie : Populus.
- génétique : Peroxidases, Populus, Protéines végétales, Superoxide dismutase.
- métabolisme : Peroxidases, Protéines végétales, Superoxide dismutase.
- méthodes : RT-PCR.
- normes : RT-PCR.
- Ascorbate peroxidases, Eau, Expression des gènes, Normes de référence, Sécheresses.
English descriptors
- KwdEn :
- Ascorbate Peroxidases (MeSH), Droughts (MeSH), Gene Expression (MeSH), Peroxidases (genetics), Peroxidases (metabolism), Plant Proteins (genetics), Plant Proteins (metabolism), Populus (enzymology), Populus (genetics), Reference Standards (MeSH), Reverse Transcriptase Polymerase Chain Reaction (methods), Reverse Transcriptase Polymerase Chain Reaction (standards), Superoxide Dismutase (genetics), Superoxide Dismutase (metabolism), Water (MeSH).
- MESH :
- chemical , genetics : Peroxidases, Plant Proteins, Superoxide Dismutase.
- chemical , metabolism : Peroxidases, Plant Proteins, Superoxide Dismutase.
- chemical : Ascorbate Peroxidases, Water.
- enzymology : Populus.
- genetics : Populus.
- methods : Reverse Transcriptase Polymerase Chain Reaction.
- standards : Reverse Transcriptase Polymerase Chain Reaction.
- Droughts, Gene Expression, Reference Standards.
Abstract
BACKGROUND
RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.
RESULTS
We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method.
CONCLUSIONS
As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.
DOI: 10.1186/1471-2199-11-57
PubMed: 20701777
PubMed Central: PMC2930637
Affiliations:
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111, 8903 Birmensdorf</wicri:regionArea><wicri:noRegion>8903 Birmensdorf</wicri:noRegion></affiliation></author><author><name sortKey="Frey, Beat" sort="Frey, Beat" uniqKey="Frey B" first="Beat" last="Frey">Beat Frey</name></author></analytic><series><title level="j">BMC molecular biology</title><idno type="eISSN">1471-2199</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Ascorbate Peroxidases (MeSH)</term><term>Droughts (MeSH)</term><term>Gene Expression (MeSH)</term><term>Peroxidases (genetics)</term><term>Peroxidases (metabolism)</term><term>Plant Proteins (genetics)</term><term>Plant Proteins (metabolism)</term><term>Populus (enzymology)</term><term>Populus (genetics)</term><term>Reference Standards (MeSH)</term><term>Reverse Transcriptase Polymerase Chain Reaction (methods)</term><term>Reverse Transcriptase Polymerase Chain Reaction (standards)</term><term>Superoxide Dismutase (genetics)</term><term>Superoxide Dismutase (metabolism)</term><term>Water (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Ascorbate peroxidases (MeSH)</term><term>Eau (MeSH)</term><term>Expression des gènes (MeSH)</term><term>Normes de référence (MeSH)</term><term>Peroxidases (génétique)</term><term>Peroxidases (métabolisme)</term><term>Populus (enzymologie)</term><term>Populus (génétique)</term><term>Protéines végétales (génétique)</term><term>Protéines végétales (métabolisme)</term><term>RT-PCR (méthodes)</term><term>RT-PCR (normes)</term><term>Superoxide dismutase (génétique)</term><term>Superoxide dismutase (métabolisme)</term><term>Sécheresses (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Peroxidases</term><term>Plant Proteins</term><term>Superoxide Dismutase</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peroxidases</term><term>Plant Proteins</term><term>Superoxide Dismutase</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Ascorbate Peroxidases</term><term>Water</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Populus</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Populus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Populus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Peroxidases</term><term>Populus</term><term>Protéines végétales</term><term>Superoxide dismutase</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Reverse Transcriptase Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Peroxidases</term><term>Protéines végétales</term><term>Superoxide dismutase</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>RT-PCR</term></keywords><keywords scheme="MESH" qualifier="normes" xml:lang="fr"><term>RT-PCR</term></keywords><keywords scheme="MESH" qualifier="standards" xml:lang="en"><term>Reverse Transcriptase Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Droughts</term><term>Gene Expression</term><term>Reference Standards</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Ascorbate peroxidases</term><term>Eau</term><term>Expression des gènes</term><term>Normes de référence</term><term>Sécheresses</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>BACKGROUND</b></p><p>RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.</p></div><div type="abstract" xml:lang="en"><p><b>RESULTS</b></p><p>We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method.</p></div><div type="abstract" xml:lang="en"><p><b>CONCLUSIONS</b></p><p>As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.</p></div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">20701777</PMID><DateCompleted><Year>2010</Year><Month>12</Month><Day>20</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1471-2199</ISSN><JournalIssue CitedMedium="Internet"><Volume>11</Volume><PubDate><Year>2010</Year><Month>Aug</Month><Day>11</Day></PubDate></JournalIssue><Title>BMC molecular biology</Title><ISOAbbreviation>BMC Mol Biol</ISOAbbreviation></Journal><ArticleTitle>Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar.</ArticleTitle><Pagination><MedlinePgn>57</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/1471-2199-11-57</ELocationID><Abstract><AbstractText Label="BACKGROUND" NlmCategory="BACKGROUND">RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.</AbstractText><AbstractText Label="RESULTS" NlmCategory="RESULTS">We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method.</AbstractText><AbstractText Label="CONCLUSIONS" NlmCategory="CONCLUSIONS">As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Regier</LastName><ForeName>Nicole</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Swiss Federal Research Institute WSL, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Frey</LastName><ForeName>Beat</ForeName><Initials>B</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D023362">Evaluation 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1;302(1):52-9</Citation><ArticleIdList><ArticleId IdType="pubmed">11846375</ArticleId></ArticleIdList></Reference></ReferenceList></PubmedData></pubmed><affiliations><list><country><li>Suisse</li></country></list><tree><noCountry><name sortKey="Frey, Beat" sort="Frey, Beat" uniqKey="Frey B" first="Beat" last="Frey">Beat Frey</name></noCountry><country name="Suisse"><noRegion><name sortKey="Regier, Nicole" sort="Regier, Nicole" uniqKey="Regier N" first="Nicole" last="Regier">Nicole Regier</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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