Validation of reference genes for quantitative real-time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real-time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real-time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical-free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol-treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate-treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real-time polymerase chain reaction-based determination of gene expression profiles in D. melanogaster treated with both chemicals.