The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
Identifieur interne : 003786 ( Main/Exploration ); précédent : 003785; suivant : 003787The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
Auteurs : Laurent Gutierrez [Suède] ; Mélanie Mauriat ; Stéphanie Guénin ; Jérôme Pelloux ; Jean-François Lefebvre ; Romain Louvet ; Christine Rusterucci ; Thomas Moritz ; François Guerineau ; Catherine Bellini ; Olivier Van WuytswinkelSource :
- Plant biotechnology journal [ 1467-7652 ] ; 2008.
Descripteurs français
- KwdFr :
- MESH :
- génétique : Arabidopsis, Gènes de plante, Populus.
- méthodes : RT-PCR.
- Analyse de profil d'expression de gènes, Reproductibilité des résultats.
English descriptors
- KwdEn :
- MESH :
- genetics : Arabidopsis, Genes, Plant, Populus.
- methods : Reverse Transcriptase Polymerase Chain Reaction.
- Gene Expression Profiling, Reproducibility of Results.
Abstract
Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
DOI: 10.1111/j.1467-7652.2008.00346.x
PubMed: 18433420
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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Thomas" sort="Moritz, Thomas" uniqKey="Moritz T" first="Thomas" last="Moritz">Thomas Moritz</name></author><author><name sortKey="Guerineau, Francois" sort="Guerineau, Francois" uniqKey="Guerineau F" first="François" last="Guerineau">François Guerineau</name></author><author><name sortKey="Bellini, Catherine" sort="Bellini, Catherine" uniqKey="Bellini C" first="Catherine" last="Bellini">Catherine Bellini</name></author><author><name sortKey="Van Wuytswinkel, Olivier" sort="Van Wuytswinkel, Olivier" uniqKey="Van Wuytswinkel O" first="Olivier" last="Van Wuytswinkel">Olivier Van Wuytswinkel</name></author></analytic><series><title level="j">Plant biotechnology journal</title><idno type="eISSN">1467-7652</idno><imprint><date when="2008" type="published">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Arabidopsis (genetics)</term><term>Gene Expression Profiling (MeSH)</term><term>Genes, Plant 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Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">18433420</PMID><DateCompleted><Year>2008</Year><Month>12</Month><Day>15</Day></DateCompleted><DateRevised><Year>2008</Year><Month>08</Month><Day>22</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1467-7652</ISSN><JournalIssue CitedMedium="Internet"><Volume>6</Volume><Issue>6</Issue><PubDate><Year>2008</Year><Month>Aug</Month></PubDate></JournalIssue><Title>Plant biotechnology journal</Title><ISOAbbreviation>Plant Biotechnol J</ISOAbbreviation></Journal><ArticleTitle>The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.</ArticleTitle><Pagination><MedlinePgn>609-18</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1111/j.1467-7652.2008.00346.x</ELocationID><Abstract><AbstractText>Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Gutierrez</LastName><ForeName>Laurent</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden. laurent.gutierrez@genfys.slu.se</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mauriat</LastName><ForeName>Mélanie</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Guénin</LastName><ForeName>Stéphanie</ForeName><Initials>S</Initials></Author><Author ValidYN="Y"><LastName>Pelloux</LastName><ForeName>Jérôme</ForeName><Initials>J</Initials></Author><Author 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IdType="doi">10.1111/j.1467-7652.2008.00346.x</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Suède</li></country></list><tree><noCountry><name sortKey="Bellini, Catherine" sort="Bellini, Catherine" uniqKey="Bellini C" first="Catherine" last="Bellini">Catherine Bellini</name><name sortKey="Guenin, Stephanie" sort="Guenin, Stephanie" uniqKey="Guenin S" first="Stéphanie" last="Guénin">Stéphanie Guénin</name><name sortKey="Guerineau, Francois" sort="Guerineau, Francois" uniqKey="Guerineau F" first="François" last="Guerineau">François Guerineau</name><name sortKey="Lefebvre, Jean Francois" sort="Lefebvre, Jean Francois" uniqKey="Lefebvre J" first="Jean-François" last="Lefebvre">Jean-François Lefebvre</name><name sortKey="Louvet, Romain" sort="Louvet, Romain" uniqKey="Louvet R" first="Romain" last="Louvet">Romain Louvet</name><name sortKey="Mauriat, Melanie" sort="Mauriat, Melanie" uniqKey="Mauriat M" first="Mélanie" last="Mauriat">Mélanie Mauriat</name><name sortKey="Moritz, Thomas" sort="Moritz, Thomas" uniqKey="Moritz T" first="Thomas" last="Moritz">Thomas Moritz</name><name sortKey="Pelloux, Jerome" sort="Pelloux, Jerome" uniqKey="Pelloux J" first="Jérôme" last="Pelloux">Jérôme Pelloux</name><name sortKey="Rusterucci, Christine" sort="Rusterucci, Christine" uniqKey="Rusterucci C" first="Christine" last="Rusterucci">Christine Rusterucci</name><name sortKey="Van Wuytswinkel, Olivier" sort="Van Wuytswinkel, Olivier" uniqKey="Van Wuytswinkel O" first="Olivier" last="Van Wuytswinkel">Olivier Van Wuytswinkel</name></noCountry><country name="Suède"><noRegion><name sortKey="Gutierrez, Laurent" sort="Gutierrez, Laurent" uniqKey="Gutierrez L" first="Laurent" last="Gutierrez">Laurent Gutierrez</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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