20 fat bodies of 5d old mutant larvae were dissected into RNAlater. The RNA was isolated using Macherey-Nagel NucleoSpin RNA II isolation Kit following the manufacturer’s protocol.
Label
Biotin
Label protocol
The quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies, Delaware, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. Total RNA samples (30 ng) were reverse-transcribed into double-stranded cDNA with Two-Cycle cDNA Synthesis Kit (Affymetrix Inc., P/N 900494, Santa Clara, CA). The double-stranded cDNA was purified using a Sample Cleanup Module (Affymetrix Inc., P/N 900371, Santa Clara, CA). The purified double-stranded cDNA were in vitro transcribed in presence of biotin-labeled nucleotides using an IVT Labeling Kit (Affymetrix Inc., P/N 900449, Santa Clara, CA). The biotinylated cRNA was purified using a Sample Cleanup Module (Affymetrix Inc., P/N 900371, Santa Clara, CA) and its quality and quantity was determined using NanoDrop ND 1000 and Bioanalyzer 2100.
Hybridization protocol
Biotin-labeled cRNA samples (15 µg) were fragmented randomly to 35–200 bp at 94°C in Fragmentation Buffer (Affymetrix Inc., P/N 900371, Santa Clara, CA) and were mixed in 300 µl of hybridization buffer containing a hybridization Control cRNA and Control Oligo B2 control (Affymetrix Inc., P/N 900454, Santa Clara, CA), 0.1 mg/ml herring sperm DNA and 0.5 mg/ml acetylated bovine serum albumin in 2-(4-morpholino)-ethane sulfonic acid (MES) buffer, pH 6.7, before hybridization to GeneChip® Drosophila Genome 2.0 Arrays for 16 h at 45°C. Arrays were then washed using an Affymetrix Fluidics Station 450 EukGE-WS2v5_450 protocol.
Scan protocol
An Affymetrix GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA) was used to measure the fluorescent intensity emitted by the labeled target.
Description
KG_02
Data processing
Data processing was performed using the Affymetrix GCOS 1.4 software (Affymetrix Inc., Santa Clara, CA). After hybridization and scanning, probe cell intensities were calculated and summarized for the respective probe sets by means of the MAS5 algorithm (Hubbell et al 2002). To compare the expression values of the genes from chip to chip, global scaling was performed, which resulted in the normalization of the trimmed mean of each chip to a target intensity (TGT value) of 500 as detailed in the statistical algorithms description document of Affymetrix (2002). Quality control measures were considered before performing the statistical analysis. These included adequate scaling factors (between 1 and 3 for all samples) and appropriate numbers of present calls calculated by application of a signed-rank call algorithm (Liu et al., 2002). The efficiency of the labeling reaction and the hybridization performance was controlled with the following parameters: Present calls and optimal 3' /5' hybridization ratios (around 1) for the housekeeping genes, for the polyA spike in controls and the prokaryotic control.
