Samples were prepared as follows. Animals were first synchronized by hypochlorite treatment and arrested at the first larval stage by incubation for 22 hours in M9. Animals were then grown at 20°C on 15 cm NG HB101 plates until the fourth larval stage (approximately 46 hours). Animals were washed, harvested in M9 and the flash frozen in liquid N2 and stored at -80° C. Total RNA was prepared by Trizol extraction.
Targets were prepared and hybridized at the Harvard Medical School Biopolymer Facility. Starting with 10 ug of total RNA first strand cDNA was synthesized as described in the Affymetrix expression technical manual.
Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 9 probe set data files were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method as implemented in RMAExpress (Bolstad/Berkeley).
