Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster
Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation.
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Subjects: | L10 - Génétique et amélioration des animaux, L50 - Physiologie et biochimie animales, Drosophila melanogaster, Transcription inverse, stress thermique, gène, http://aims.fao.org/aos/agrovoc/c_30543, http://aims.fao.org/aos/agrovoc/c_35129, http://aims.fao.org/aos/agrovoc/c_11488, http://aims.fao.org/aos/agrovoc/c_3214, |
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dig-cirad-fr-5619392024-01-28T19:44:00Z http://agritrop.cirad.fr/561939/ http://agritrop.cirad.fr/561939/ Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. Ponton Fleur, Chapuis Marie-Pierre, Pernice Mathieu, Sword Gregory A., Simpson Stephen J.. 2011. Journal of Insect Physiology, 57 (6) : 840-850.https://doi.org/10.1016/j.jinsphys.2011.03.014 <https://doi.org/10.1016/j.jinsphys.2011.03.014> Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster Ponton, Fleur Chapuis, Marie-Pierre Pernice, Mathieu Sword, Gregory A. Simpson, Stephen J. eng 2011 Journal of Insect Physiology L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation. article info:eu-repo/semantics/article Journal Article info:eu-repo/semantics/publishedVersion http://agritrop.cirad.fr/561939/1/document_561939.pdf application/pdf Cirad license info:eu-repo/semantics/restrictedAccess https://agritrop.cirad.fr/mention_legale.html https://doi.org/10.1016/j.jinsphys.2011.03.014 10.1016/j.jinsphys.2011.03.014 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jinsphys.2011.03.014 info:eu-repo/semantics/altIdentifier/purl/https://doi.org/10.1016/j.jinsphys.2011.03.014 |
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L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 |
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L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 Ponton, Fleur Chapuis, Marie-Pierre Pernice, Mathieu Sword, Gregory A. Simpson, Stephen J. Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
description |
Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation. |
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article |
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L10 - Génétique et amélioration des animaux L50 - Physiologie et biochimie animales Drosophila melanogaster Transcription inverse stress thermique gène http://aims.fao.org/aos/agrovoc/c_30543 http://aims.fao.org/aos/agrovoc/c_35129 http://aims.fao.org/aos/agrovoc/c_11488 http://aims.fao.org/aos/agrovoc/c_3214 |
author |
Ponton, Fleur Chapuis, Marie-Pierre Pernice, Mathieu Sword, Gregory A. Simpson, Stephen J. |
author_facet |
Ponton, Fleur Chapuis, Marie-Pierre Pernice, Mathieu Sword, Gregory A. Simpson, Stephen J. |
author_sort |
Ponton, Fleur |
title |
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
title_short |
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
title_full |
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
title_fullStr |
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
title_full_unstemmed |
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster |
title_sort |
evaluation of potential reference genes for reverse transcription-qpcr studies of physiological responses in drosophila melanogaster |
url |
http://agritrop.cirad.fr/561939/ http://agritrop.cirad.fr/561939/1/document_561939.pdf |
work_keys_str_mv |
AT pontonfleur evaluationofpotentialreferencegenesforreversetranscriptionqpcrstudiesofphysiologicalresponsesindrosophilamelanogaster AT chapuismariepierre evaluationofpotentialreferencegenesforreversetranscriptionqpcrstudiesofphysiologicalresponsesindrosophilamelanogaster AT pernicemathieu evaluationofpotentialreferencegenesforreversetranscriptionqpcrstudiesofphysiologicalresponsesindrosophilamelanogaster AT swordgregorya evaluationofpotentialreferencegenesforreversetranscriptionqpcrstudiesofphysiologicalresponsesindrosophilamelanogaster AT simpsonstephenj evaluationofpotentialreferencegenesforreversetranscriptionqpcrstudiesofphysiologicalresponsesindrosophilamelanogaster |
_version_ |
1792497971727695872 |