normalization data transformation protocol
Data normalisation was performed using the Microarray Suite version 5 (MAS 5.0) statistical algorithm using the Affymetrix Expression Console software. Probes where the detection p-value (calculated using the intensity value of a perfect match to a mismatch sequence) was >0.062 in any of the samples were classed as absent and excluded from further analysis. ID_REF = VALUE = MAS5.0 signal intensity Detection = Detection p-value = Pairs =
array scanning protocol
Imaging of the arrays was performed using the Affymetrix GCS3000 microarray system.
Hybridisations were performed using the Genechip 3’IVT kit (Affymetrix) on Genechip Drosophila Genome 2.0 Arrays (Affymetrix).
cRNA was prepared from 500ng of total RNA using the Ambion Premier kit (Ambion).
Larvae were grown on standard fly food at 25 degrees.
sample treatment protocol
One control and two different experimental genotypes were used: Control (repoGal4/+), Htl[ACT] (repoGal4>UAS-Htl[ACT]) and InR (repoGal4>UAS-InR).
nucleic acid extraction protocol
For the microarray analysis the complete CNS from 10-15 wandering third instar larvae were dissected in PBS and placed into PBS on ice and then transferred into 100ul of lysis buffer from the Absolutely RNA Microprep kit (Stratagene) and vortexed for 5 seconds. Total RNA was then prepared using this kit. For each genotype RNA samples were prepared in triplicate and stored at -80oC.