8 micron cryosections were transferred onto PALM membrane slides (P.A.L.M. Microlaser Technologies) and air dried on ice for about 1 minute. Slides were immersed for 2 minutes in cold 75% ethanol, tapped dry and stained for 30 seconds in cold haematoxylin and eosin (9:1) containing 1% NucleoGuard (AmpTec, Hamburg). Excess stain was tapped off and slides were washed in cold nuclease-free water for 30 seconds, cold 75% ethanol for 1 minute, cold 100% ethanol for 1 minute then air dried. Tumour cells for analysis were excised by laser microdissection and pressure catapulting using a PALM MicroBeam instrument and caught on PALM Adhesive Caps. A minimum of 200,000 µm2 of tissue was collected for each DNA extraction. DNA was extracted from microdissected cells by adding 100 µl of lysis buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA, 1% Tween 20, 0.4 mg/ml proteinase K (Qiagen)) to the tube containing the captured cells and incubating inverted for 3hr at 55OC then 5min at 95 OC. 2 µl of linear polyacrylamide solution (GenElute, Sigma) were added and DNA was recovered by ethanol precipitation. The pellet was washed with 70% ethanol and air dried. The precipitated DNA was subject to whole genome amplification using a Genomiphi kit (GE Healthcare) according to the manufacturer’s instructions.
Label
biotin
Label protocol
DNA was labelled according to Affymetrix standard protocol.
Hybridization protocol
All arrays were washed and stained on an Affymetrix FS450 fluidics station.
Scan protocol
All arrays were scanned using an Affymetrix GeneChip 3000 7G scanner as per Affymetrix procedures.
Data processing
Genotype analysis was performed using Affymetrix Genotyping Console version 4.0 with the default settings.
