Evaluation of reference genes for insect olfaction studies
- Autores
- Omondi, Bonaventure Aman; Latorre Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo
- Año de publicación
- 2015
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- 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.
Fil: Omondi, Bonaventure Aman. No especifíca;
Fil: Latorre Estivalis, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina
Fil: Rocha Oliveira, Ivana Helena. No especifíca;
Fil: Ignell, Rickard. No especifíca;
Fil: Lorenzo, Marcelo Gustavo. No especifíca; - Materia
-
NORMALIZATION PROCESS
OLFACTION AND TRIATOMINES
REFERENCE GENES
RT-QPCR - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/210333
Ver los metadatos del registro completo
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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 Técnicas2025-09-03T10:11:30Zoai: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:34982025-09-03 10:11:31.011CONICET 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 |
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 |
topic |
NORMALIZATION PROCESS OLFACTION AND TRIATOMINES REFERENCE GENES RT-QPCR |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
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. Fil: Omondi, Bonaventure Aman. No especifíca; Fil: Latorre Estivalis, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Rocha Oliveira, Ivana Helena. No especifíca; Fil: Ignell, Rickard. No especifíca; Fil: Lorenzo, Marcelo Gustavo. No especifíca; |
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 |
reponame_str |
CONICET Digital (CONICET) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
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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1842270161271259136 |
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13.13397 |