Total RNA was extracted from the human pituitary tumours using Trizol (Invitrogen, Carlsbad, California, USA), according to the manufacturer’s protocol.
Label
biotin
Label protocol
Total RNA (2 µg) was amplified and biotin-labelled by a round of in vitro transcription (IVT) with a Message Amp aRNA kit (Ambion, Austin, Texas, USA) following the manufacturer’s protocol.
Hybridization protocol
Ten micrograms of biotin-labeled aRNA was fragmented using 5 μl of fragmentation buffer in a final volume of 20 µl then was mixed with 240 µl of Amersham hybridization solution (GE Healthcare Europe GmbH, Freiburg, Germany) and injected onto CodeLink Uniset Human Whole Genome bioarrays (GE Healthcare Europe GmbH, Freiburg, Germany). Arrays were hybridized overnight at 37°C at 300 rpm in an incubator. The slides were washed in stringent TNT buffer at 46°C for 1 hour, then a streptavidin-cy5 (GE Healthcare) detection step was performed. Each slide was incubated for 30 min in 3.4 ml of streptavidin-cy5 solution as described previously (Fevre-Montange et al 2006), then was washed 4 times in 240 ml of TNT buffer, rinsed twice in 240 ml of water containing 0.2%Triton X-100, and dried by centrifugation at 600 rpm.
Scan protocol
The slides were scanned using a GenePix 4000B scanner (Axon, Union City, USA) and GenePix 3.0 software, with the laser set at 635 mm, the laser power at 100%, and the photomultiplier tube voltage at 60%.
Data processing
The relative intensity of the raw hybridization signal on arrays varies in different experiments. CodeLink Expression Analysis v4.0 software was therefore used to normalize the raw hybridization signal on each array to the median of the array (median intensity is 1 after normalization) for better cross-array comparison. The threshold of detection was calculated using the normalized signal intensity of the 100 negative control probes in the array; spots with signal intensities below this threshold are referred to as “absent”.
