Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.
Identifieur interne : 000369 ( Main/Exploration ); précédent : 000368; suivant : 000370Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.
Auteurs : Marcos Paolinelli-Alfonso [Mexique] ; Clara Elizabeth Galindo-Sánchez [Mexique] ; Rufina Hernandez-Martinez [Mexique]Source :
- Journal of microbiological methods [ 1872-8359 ] ; 2016.
Descripteurs français
- KwdFr :
- Analyse de profil d'expression de gènes (méthodes), Ascomycota (génétique), Glyceraldehyde 3-phosphate dehydrogenases (génétique), Gènes fongiques (MeSH), Maladies des plantes (microbiologie), Réaction de polymérisation en chaine en temps réel (méthodes), Tubuline (génétique), Vitis (microbiologie).
- MESH :
- génétique : Ascomycota, Glyceraldehyde 3-phosphate dehydrogenases, Tubuline.
- microbiologie : Maladies des plantes, Vitis.
- méthodes : Analyse de profil d'expression de gènes, Réaction de polymérisation en chaine en temps réel.
- Gènes fongiques.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Glyceraldehyde-3-Phosphate Dehydrogenases, Tubulin.
- genetics : Ascomycota.
- methods : Gene Expression Profiling, Real-Time Polymerase Chain Reaction.
- microbiology : Plant Diseases, Vitis.
- Genes, Fungal.
Abstract
Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.
DOI: 10.1016/j.mimet.2016.05.021
PubMed: 27237774
Affiliations:
Links toward previous steps (curation, corpus...)
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It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">27237774</PMID><DateCompleted><Year>2017</Year><Month>08</Month><Day>25</Day></DateCompleted><DateRevised><Year>2018</Year><Month>04</Month><Day>30</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1872-8359</ISSN><JournalIssue CitedMedium="Internet"><Volume>127</Volume><PubDate><Year>2016</Year><Month>08</Month></PubDate></JournalIssue><Title>Journal of microbiological methods</Title><ISOAbbreviation>J Microbiol Methods</ISOAbbreviation></Journal><ArticleTitle>Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.</ArticleTitle><Pagination><MedlinePgn>82-88</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S0167-7012(16)30108-7</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.mimet.2016.05.021</ELocationID><Abstract><AbstractText>Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.</AbstractText><CopyrightInformation>Copyright © 2016 Elsevier B.V. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Paolinelli-Alfonso</LastName><ForeName>Marcos</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. Electronic address: marcos_paolinelli@yahoo.com.ar.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Galindo-Sánchez</LastName><ForeName>Clara Elizabeth</ForeName><Initials>CE</Initials><AffiliationInfo><Affiliation>Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. Electronic address: cgalindo@cicese.mx.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hernandez-Martinez</LastName><ForeName>Rufina</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. 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