Microarray analysis of gene expression was carried out on the Berlin genetic background strain. For each treatment condition, 3 groups of 200 male flies each were treated identically and combined prior to RNA extraction; this design helped to minimize variations in ethanol dose and time of individual experiments. Treatment conditions were exposure to either humidified air (flow rate: 3.7 l/min) or ethanol vapor (saturated ethanol vapor diluted to 60% with humidified air to 3.7 l/min) for 30 minutes at 25°C in perforated 50 ml conical tubes. Treated flies were allowed to recover in standard culturing conditions (food containing vials kept at 25°C and 70% humidity) for 0, 30, 60, 90, 120, 180, and 210 minutes. Additionally, 2 independent groups of flies were given no treatment. Flies were quickly frozen and heads were isolated. RNA from the resulting 16 treatment conditions was hybridized to Affymetrix Drosophila 2.0 oligonucleotide microarray chips at the Partners HealthCare Center for Personalized Genetic Medicine Microarray Facility (Harvard University).
