Evaluation of reference genes for insect olfaction studies

Background: Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting mate...

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Autores: Omondi, Bonaventure Aman, Latorre Estivalis, Jose Manuel, Rocha Oliveira, Ivana Helena, Ignell, Rickard, Lorenzo, Marcelo Gustavo
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2015
País:Argentina
Institución:Consejo Nacional de Investigaciones Científicas y Técnicas
Repositorio:CONICET Digital (CONICET)
Idioma:inglés
OAI Identifier:oai:ri.conicet.gov.ar:11336/210333
Acceso en línea:http://hdl.handle.net/11336/210333
Access Level:acceso abierto
Palabra clave:NORMALIZATION PROCESS
OLFACTION AND TRIATOMINES
REFERENCE GENES
RT-QPCR
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
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spelling Evaluation of reference genes for insect olfaction studiesOmondi, Bonaventure AmanLatorre Estivalis, Jose ManuelRocha Oliveira, Ivana HelenaIgnell, RickardLorenzo, Marcelo GustavoNORMALIZATION PROCESSOLFACTION AND TRIATOMINESREFERENCE GENESRT-QPCRhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Background: Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. Methods: We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes. Results: We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards. Conclusions: This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.Fil: Omondi, Bonaventure Aman. No especifíca;Fil: Latorre Estivalis, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Rocha Oliveira, Ivana Helena. No especifíca;Fil: Ignell, Rickard. No especifíca;Fil: Lorenzo, Marcelo Gustavo. No especifíca;BioMed Central2015-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/210333Omondi, Bonaventure Aman; Latorre Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo; Evaluation of reference genes for insect olfaction studies; BioMed Central; Parasites and Vectors; 8; 1; 4-2015; 1-151756-3305CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-015-0862-xinfo:eu-repo/semantics/altIdentifier/doi/10.1186/s13071-015-0862-xinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2024-05-08T14:27:50Zoai:ri.conicet.gov.ar:11336/210333instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982024-05-08 14:27:50.667CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Evaluation of reference genes for insect olfaction studies
title Evaluation of reference genes for insect olfaction studies
spellingShingle Evaluation of reference genes for insect olfaction studies
Omondi, Bonaventure Aman
NORMALIZATION PROCESS
OLFACTION AND TRIATOMINES
REFERENCE GENES
RT-QPCR
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
title_short Evaluation of reference genes for insect olfaction studies
title_full Evaluation of reference genes for insect olfaction studies
title_fullStr Evaluation of reference genes for insect olfaction studies
title_full_unstemmed Evaluation of reference genes for insect olfaction studies
title_sort Evaluation of reference genes for insect olfaction studies
dc.creator.none.fl_str_mv Omondi, Bonaventure Aman
Latorre Estivalis, Jose Manuel
Rocha Oliveira, Ivana Helena
Ignell, Rickard
Lorenzo, Marcelo Gustavo
author Omondi, Bonaventure Aman
author_facet Omondi, Bonaventure Aman
Latorre Estivalis, Jose Manuel
Rocha Oliveira, Ivana Helena
Ignell, Rickard
Lorenzo, Marcelo Gustavo
author_role author
author2 Latorre Estivalis, Jose Manuel
Rocha Oliveira, Ivana Helena
Ignell, Rickard
Lorenzo, Marcelo Gustavo
author2_role author
author
author
author
dc.subject.none.fl_str_mv NORMALIZATION PROCESS
OLFACTION AND TRIATOMINES
REFERENCE GENES
RT-QPCR
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
topic NORMALIZATION PROCESS
OLFACTION AND TRIATOMINES
REFERENCE GENES
RT-QPCR
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
description Background: Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. Methods: We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes. Results: We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards. Conclusions: This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.
publishDate 2015
dc.date.none.fl_str_mv 2015-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/210333
Omondi, Bonaventure Aman; Latorre Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo; Evaluation of reference genes for insect olfaction studies; BioMed Central; Parasites and Vectors; 8; 1; 4-2015; 1-15
1756-3305
CONICET Digital
CONICET
url http://hdl.handle.net/11336/210333
identifier_str_mv Omondi, Bonaventure Aman; Latorre Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo; Evaluation of reference genes for insect olfaction studies; BioMed Central; Parasites and Vectors; 8; 1; 4-2015; 1-15
1756-3305
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-015-0862-x
info:eu-repo/semantics/altIdentifier/doi/10.1186/s13071-015-0862-x
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 15.811543

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