Prior to hybridisation, the fragmented cDNA was heated to 95°C for 5 min and subsequently to 45°C for 5 min before loading onto the Affymetrix Human Exon 1.0 ST array cartridge. The array cartridge was then incubated for 16 h at 45°C at constant rotation (60 rpm). The washing and staining procedure was performed in the Affymetrix Fluidics Station 450. The array was exposed to 10 washes in 6× SSPE-T at 250°C followed by four washes in 0.5× SSPE-T at 50°C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, final concentration 2 mg/mL (Affymetrix) in 6× saline sodium phosphate EDTA buffer with 0.01% Tween-20 (SSPE-T) for 30 min at 25°C followed by 10 washes in 6× SSPE-T at 25°C. This was followed by an antibody amplification step using normal goat IgG as blocking reagent, final concentration 0.1 mg/mL (Affymetrix) and biotinylated anti-streptavidin antibody (goat), final concentration 3 mg/mL (Affymetrix). This was followed by a staining step with a streptavidin–phycoerythrin conjugate, final concentration 2 mg/mL (Affymetrix, UK) in 6× SSPE-T for 30 min at 25°C and 10 washes in 6× SSPE-T at 25°C. The arrays were scanned at 560 nm using a confocal laser-scanning microscope (GeneChip® Scanner 3000 7G).
