Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

Detalhes bibliográficos
Autor(a) principal: Barsalobres-Cavallari, Carla F. [UNESP]
Data de Publicação: 2009
Outros Autores: Severino, Fabio E. [UNESP], Maluf, Mirian P., Maia, Ivan de Godoy [UNESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.1186/1471-2199-10-1
http://hdl.handle.net/11449/18013
Resumo: Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.
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spelling Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditionsBackground: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.UNESP, Inst Biociencias, Dept Genet, BR-18618000 Botucatu, SP, BrazilCtr Cafe Alcides Carvalho, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) IAC, São Paulo, BrazilUNESP, Inst Biociencias, Dept Genet, BR-18618000 Botucatu, SP, BrazilBiomed Central Ltd.Universidade Estadual Paulista (Unesp)Ctr Cafe Alcides CarvalhoBarsalobres-Cavallari, Carla F. [UNESP]Severino, Fabio E. [UNESP]Maluf, Mirian P.Maia, Ivan de Godoy [UNESP]2014-05-20T13:50:28Z2014-05-20T13:50:28Z2009-01-06info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article11application/pdfhttp://dx.doi.org/10.1186/1471-2199-10-1Bmc Molecular Biology. London: Biomed Central Ltd., v. 10, p. 11, 2009.1471-2199http://hdl.handle.net/11449/1801310.1186/1471-2199-10-1WOS:000263177900001WOS000263177900001.pdf8649222099176162Web of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengBmc Molecular Biology2.7951,216info:eu-repo/semantics/openAccess2023-12-24T06:15:36Zoai:repositorio.unesp.br:11449/18013Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-12-24T06:15:36Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
title Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
spellingShingle Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
Barsalobres-Cavallari, Carla F. [UNESP]
title_short Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
title_full Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
title_fullStr Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
title_full_unstemmed Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
title_sort Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
author Barsalobres-Cavallari, Carla F. [UNESP]
author_facet Barsalobres-Cavallari, Carla F. [UNESP]
Severino, Fabio E. [UNESP]
Maluf, Mirian P.
Maia, Ivan de Godoy [UNESP]
author_role author
author2 Severino, Fabio E. [UNESP]
Maluf, Mirian P.
Maia, Ivan de Godoy [UNESP]
author2_role author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (Unesp)
Ctr Cafe Alcides Carvalho
dc.contributor.author.fl_str_mv Barsalobres-Cavallari, Carla F. [UNESP]
Severino, Fabio E. [UNESP]
Maluf, Mirian P.
Maia, Ivan de Godoy [UNESP]
description Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.
publishDate 2009
dc.date.none.fl_str_mv 2009-01-06
2014-05-20T13:50:28Z
2014-05-20T13:50:28Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
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format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1186/1471-2199-10-1
Bmc Molecular Biology. London: Biomed Central Ltd., v. 10, p. 11, 2009.
1471-2199
http://hdl.handle.net/11449/18013
10.1186/1471-2199-10-1
WOS:000263177900001
WOS000263177900001.pdf
8649222099176162
url http://dx.doi.org/10.1186/1471-2199-10-1
http://hdl.handle.net/11449/18013
identifier_str_mv Bmc Molecular Biology. London: Biomed Central Ltd., v. 10, p. 11, 2009.
1471-2199
10.1186/1471-2199-10-1
WOS:000263177900001
WOS000263177900001.pdf
8649222099176162
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Bmc Molecular Biology
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dc.format.none.fl_str_mv 11
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dc.publisher.none.fl_str_mv Biomed Central Ltd.
publisher.none.fl_str_mv Biomed Central Ltd.
dc.source.none.fl_str_mv Web of Science
reponame:Repositório Institucional da UNESP
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