Groups of 50-60 ca. one week old flies were allowed to lay eggs for three hours on apple juice agar medium (100 ml apple juice, 300 ml water, 6 g sucrose, 3.6 g agar) dispensed in 5 ml aliquots into the lids of 35 mm diameter Petri dishes. Each laying cap was then placed in a 60 mm Petri dish, to which 10 ml of either 18% ethanol (“exposure E”) or distilled water (“exposure W”) was added, with the result that the liquid just covered (by 1 mm or so) the surface of the agar (18% ethanol was used to result in an approximate final concentration of 12%, taking into account the volume of laying medium). The dishes were covered, sealed with Parafilm, and placed on an orbital shaker at a gentle setting for 15 hours. After this period, the caps were removed from the dishes and rinsed with distilled water, to remove any early hatching larvae. Thereafter, newly-hatched larvae were collected at 45 minute intervals.
Growth protocol
To control for environmental effects on gene expression, flies from all populations were reared for at least two generations on standard medium at 25ºC before being used to lay eggs.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from groups of 50-200 larvae using the RNeasy mini kit and QIAshredder columns (QIAGEN Inc., Valencia, CA U.S.A.). Extractions were conducted in three blocks on different days, at the same time each day to minimize circadian effects, with one sample per population and pre-treatment per block. One RNA sample was lost due to technical error, leaving 35 samples for microarray hybridization.
Label
biotin
Label protocol
Total RNA was converted to biotin-labeled, fragmented cDNA with kits from NuGEN Technologies (Ovation Amplification System V2, FL-Ovation cDNA Biotin Module 2).
Hybridization protocol
The cDNA was hybridized overnight to Drosophila Genome 2.0 arrays (Affymetrix, Santa Clara, CA U.S.A.), which were washed and stained with a fluorescent dye that binds to biotin (streptavidin, R-phycoerythrin conjugate).
Scan protocol
Arrays were scanned as recommended by Affymetrix using an Affymetrix Fluidics Station 450 and Scanner 3000.
Data processing
Raw expression .CEL files were normalized using the gcRMA algorithm (Wu et al. 2004) as implemented in the Affy package on Bioconductor (version 2.4) (Gentleman et al. 2004). We filtered out probe sets with low expression and variance by removing those whose mean, interquartile range among arrays, and standard deviation among arrays were each less than the median of the respective quantity over all probesets. After the filtering step, 12,236 out of the 18,769 original probe sets were left for further analysis.
