Drosophila stocks and crosses. Female flies originated from a highly inbred, wild-type Canton S stock. These females wer mated to males that do not produce sperm or accessory gland proteins (Acps). Males deficient in the production of sperm and Acps (DTA-E) were derived from a transgenic stock with a Canton S background. DTA-E males do not produce sperm and do not make Acps from the main cells of their accessory glands (96% of the secretory cells of this tissue).
Flies were reared on standard yeast/glucose medium at 25oC under a 12:12 hour light:dark cycle, collected as virgins and aged three to four days prior to experimental use. Within three hours of the beginning of their light cycle, single virgin females were paired with single virgin males of the appropriate genotype and allowed to mate. Mated female flies were flash-frozen in liquid nitrogen between one and three hours after the completion of mating and stored at -80oC until extraction of total RNA.
Sample preparation. Frozen flies from replicate treatments were pooled to minimize potential variation in gene expression from time and/or day of mating. From each pooled batch, 50 flies were randomly chosen and separated into two equal batches for RNA extraction. Total RNA was prepared using Trizol (Invitrogen, Inc.) extraction and purified with RNeasy Mini kit (Qiagen). According to the manufacturer’s instructions, cDNA was prepared from the samples and hybridized to Drosophila Affymetrix GeneChips (Affymetrix, Santa Clara, CA) by the University of Kentucky Microarray Core Facility (Lexington, KY). Each experimental treatment consisted of two independent RNA extractions each replicated twice, for a total of four chips per treatment.
Hybridization conditions: Rotating for 16 hours at 45 degrees C.
