Full length articleEvaluation of reference genes for gene expression studies using quantitative real-time PCR in Drosophila melanogaster after chemical exposures
Introduction
The fruit fly, Drosophila melanogaster, has been widely used as an important model organism in various areas of biological science (Jennings, 2011). D. melanogaster is attracted to fermentation-associated volatile chemicals, such as ethanol, acetic acid, ethyl acetate, acetaldehyde, and methanol, which are mainly found in overripe, decaying, abandoned, or fermented fruits (Zhu et al., 2003). Considering this distinctive habitat, defined by the presence of environmentally toxic chemicals, D. melanogaster is thought to have adapted by evolving tolerance to these chemicals. We recently discovered that D. melanogaster exhibited higher tolerance to toxic chemicals such as ethanol, acetic acid, and 2-phenylethanol than that exhibited by Drosophila suzukii which prefers fresh fruits (Kim et al., 2018). According to previous studies, a high frequency of alcohol dehydrogenase (adh) alleles and induced expression of adh have been suggested to be the underlying reasons for D. melanogaster tolerance to ethanol and acetic acid (Devineni and Heberlein, 2009, Montooth et al., 2006, Ogueta et al., 2010). Increased glutathione-S-transferase (GST) activity and GST gene numbers are found in D. melanogaster than in D. suzukii, suggesting an enhanced tolerance of the former to chemical-induced oxidative stress during fermentation and overripening (Nguyen et al., 2016). A more recent study demonstrated that D. melanogaster is more tolerant to the accumulation of nitrogen waste, such as ammonia and urea, produced by metabolic activity of microorganisms during fermentation, owing to increased expression or activity of ornithine aminotransferase and GST, both of which are known to be involved in nitrogen metabolism (Belloni et al., 2018).
Identification of genes involved in chemical detoxification or metabolism and investigation of their expression patterns are essential for understanding the evolutionary adaptation of D. melanogaster to its distinctive habitat. Quantitative real-time PCR (qRT-PCR) is a fast, sensitive, replicable, and accurate method that has been widely used to analyze the expression patterns of putative genes associated with chemical adaptation in D. melanogaster (Ling and Salvaterra, 2011, Zhai et al., 2014). However, several studies have revealed that confounding variations may exist from sample to sample and run to run, usually resulting from different treatments, RNA extraction techniques, or amplification efficiency (Ling and Salvaterra, 2011, Zhai et al., 2014). Therefore, data normalization using internal reference genes is a crucial step to compensate for sample variations. Therefore, reference genes should be stably expressed across different experimental conditions (Zhai et al., 2014) to facilitate accurate comparisons between target gene expression patterns under chemical stress. Despite the importance of D. melanogaster as a model organism in gene expression studies of physiological responses, the systematic validation of reference genes for qRT-PCR has received little attention, and qRT-PCR is still being carried out without reference gene validation in many studies (Ponton et al., 2011).
Considering importance of reference gene selection for qRT-PCR and environmental adaptation of D. melanogaster to various chemicals, we recently validated the expression stability of five reference genes in the fly treated with methanol and ethyl acetate (Kim et al., 2019). In addition to our recent study, in the present study, we aimed to identify the most suitable reference genes for quantifying target gene expression using qRT-PCR in D. melanogaster exposed to various concentration of three chemicals (acetic acid, ethanol, and 2-phenylethanol) majorly produced from overripe, decaying, abandoned, and fermented environment. To accomplish this, we selected ten candidate reference genes, including two ribosomal proteins (rpL18 and rpS3), heat shock protein 22 (hsp22), elongation factor 1 β (ef1β), TATA box-binding protein (tbp), glyceraldehyde-3-phosphate dehydrogenase 1 (gapdh), acetylcholinesterase (ace), arginine kinase (argk), NADH dehydrogenase (nd), and α-tubulin (α-tub) by referring to the previous studies with modification (Kim et al., 2019, Zhai et al., 2014), and their expression stabilities were examine. The suitability of each of these genes as references was analyzed using three different software platforms: geNorm, NormFinder, and BestKeeper. We also validated the normalization effect of the selected reference genes against the expression patterns of adh, which has been known to play a crucial role in ethanol tolerance of D. melanogaster (Devineni and Heberlein, 2009, Montooth et al., 2006, Ogueta et al., 2010).
Section snippets
Insects and sample preparation
The wild-type strain Canton-S of D. melanogaster initially originated from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA) to the Ilsong Institute of Life Science, Hallym University (Anyang, Gyeonggi-do, Republic of Korea). We then procured the D. melanogaster used in this study from the Ilsong Institute of Life Science (Kim et al., 2018, Kim et al., 2014). This organism was maintained at 25 ± 2 °C, 16 L:8 D photoperiod, and 50%–70% relative humidity according
Amplification specificity and efficiency
The primer specificity and amplification efficiency of candidate reference genes for qRT-PCR were checked before their expression stability was analyzed. The PCR products of ten potential reference genes were amplified with specifically designed primer sets (Table 1). Gene-specific amplification of all ten genes was confirmed by visualizing a single amplicon at the expected size (Fig. S1) on a 2% agarose gel and by observing a single peak during melting temperature analysis in real-time PCR
Discussion
D. melanogaster is a frequently used experimental model to understand the physiological functions of specific genes because of its short life cycle, high reproductivity, and ease of culture in laboratories (Jennings, 2011, Ponton et al., 2011). Because the preferred habitat of this organism is rotten and fermented fruits (Zhu et al., 2003), it also exhibits a high tolerance for chemicals produced during the fermentation process (Kim et al., 2018), D. melanogaster is also used as a model to
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgments
We thank Dr. Young Ho Kho (Hallym University) for providing the strain of D. melanogaster. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1C1B2008699).
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