Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster

Ponton, F; Chapuis, MP; Pernice, M; Sword, GA; Simpson, SJ

Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster

Ponton, F Chapuis, MP Pernice, M

Sword, GA Simpson, SJ

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Publication Type:: Journal Article
Citation:: Journal of Insect Physiology, 2011, 57 (6), pp. 840 - 850
Issue Date:: 2011-06-01

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Field	Value	Language
dc.contributor.author	Ponton, F	en_US
dc.contributor.author	Chapuis, MP	en_US
dc.contributor.author	Pernice, M https://orcid.org/0000-0002-3431-2104	en_US
dc.contributor.author	Sword, GA	en_US
dc.contributor.author	Simpson, SJ	en_US
dc.date.available	2011-03-15	en_US
dc.date.issued	2011-06-01	en_US
dc.identifier.citation	Journal of Insect Physiology, 2011, 57 (6), pp. 840 - 850	en_US
dc.identifier.issn	0022-1910	en_US
dc.identifier.uri	http://hdl.handle.net/10453/32414
dc.description.abstract	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. © 2011.	en_US
dc.relation.ispartof	Journal of Insect Physiology	en_US
dc.relation.isbasedon	10.1016/j.jinsphys.2011.03.014	en_US
dc.subject.classification	Entomology	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Drosophila melanogaster	en_US
dc.subject.mesh	Drosophila Proteins	en_US
dc.subject.mesh	Gene Expression Profiling	en_US
dc.subject.mesh	Reverse Transcriptase Polymerase Chain Reaction	en_US
dc.subject.mesh	Reference Standards	en_US
dc.title	Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster	en_US
dc.type	Journal Article
utslib.citation.volume	6	en_US
utslib.citation.volume	57	en_US
utslib.for	0604 Genetics	en_US
utslib.for	0606 Physiology	en_US
utslib.for	0608 Zoology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - C3 - Climate Change Cluster
utslib.copyright.status	closed_access
pubs.issue	6	en_US
pubs.publication-status	Published	en_US
pubs.volume	57	en_US

Abstract:

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. © 2011.

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http://hdl.handle.net/10453/32414