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

Fleur Ponton*, Marie Pierre Chapuis, Mathieu Pernice, Gregory A. Sword, Stephen J. Simpson

*Corresponding author for this work

Research output: Contribution to journalArticle

182 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)840-850
Number of pages11
JournalJournal of Insect Physiology
Volume57
Issue number6
DOIs
Publication statusPublished - Jun 2011
Externally publishedYes

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