array scanning and feature extraction protocol
P-AFFY-6 Affymetrix CEL analysis
Affymetrix Generic Hybridization
Data were GC-RMA normalized with Bioconductor and the R project for statistical computing (Wu & Irizarry, 2004), using the BRB-Tools program developed at the Biometric Research Branch of the Division of Cancer Treatment & Diagnosis of the National Cancer Institute (Maryland, USA).
RNA processing, hybridization and scanning were performed at the Laboratory of Molecular Oncology, HUCA (Asturias, Spain) according the manufacturer's instructions, from 3ug total RNA for each of the 8 samples.
In the experimental group 2-8 days old flies were placed 48h at 30 +/- 1 degree C. For the control group, flies remained at growing temperature. Prior treatment experimental and control flies were transferred to new bottles with a near odorless medium composed by 5g/l agarose, 50g/l sucrose and water. With this measure we try to minimize the differential effect of environmental odor in the experimental group versus control. Third antennal segments were obtained by freezing flies in liquid nitrogen, breakage and collection. A high number of samples, approximately 4000 third antennal segments for each replicate, were collected to achieve a sufficient representation of genes with low expression levels that could be missing with other protocols. Each replicate contained 2 groups of 2000 segments collected from 11:00 am to 14:00 pm and 16:00 pm to 19:00 pm, respectively, to prevent misassignments of gene expression due to circadian fluctuations.
The standard line of Drosophila melanogaster Canton-S, obtained from the Bloomington Stock Center (BSC, Indiana, USA) was used. Flies were bred at 21 +/1 degree C, with light/dark cycles of 12:12 hours, in 220 cc. bottles with a common yeast-sugar-agar medium.
Total RNA was purified with Nucleospin RNA II kit (Macherey-Nagel), following manufacturer's instructions.