Pulverized tissue was lysed in 200mM NaCl, 5mM EDTA, 0.2% SDS, 100mM Tris pH8.5, 100ug/ml proteinase K overnight at 55C, then extracted with phenol/chloroform.
Label
Cy5
Label protocol
Genomic DNA Labeling Kit (Enzo Life Sciences, Farmingdale, NY) was used as per manufacturer's protocol.
Pulverized tissue was lysed in 200mM NaCl, 5mM EDTA, 0.2% SDS, 100mM Tris pH8.5, 100ug/ml proteinase K overnight at 55C, then extracted with phenol/chloroform.
Label
Cy3
Label protocol
Genomic DNA Labeling Kit (Enzo Life Sciences, Farmingdale, NY) was used as per manufacturer's protocol.
Hybridization protocol
Prior to hybridization, the test and reference probes were combined with 100 ug mouse fluorimetric Cot-1 (Invitrogen) and ethanol precipitated. Following centrifugation, the probes were resuspended in 110ul SlideHyb Buffer #3 (Ambion) containing 5ul of 100µg/ul Yeast tRNA (Invitrogen), heated to 95ºC for 5 minutes and incubated for 30 minutes at 37ºC to block repetitive elements on the probe. Hybridization was performed for 16 hours at 55ºC using a GeneMachine hybridization station (Genomic Solutions, Inc.) as described (Cowell, J. K. and N. J. Nowak. 2003. High-resolution analysis of genetic events in cancer cells using bacterial artificial chromosome arrays and comparative genome hybridization. Adv Cancer Res 90:91-125). After hybridization, the slides were automatically washed in the GeneMachines station with reducing concentrations of SSC and SDS. The final wash was 30 seconds in 0.1X SSC, followed by a two second 100 % ethanol dip. The slides were spun dry and scanned immediately at 5µm resolution using a GenePix 4000B laser scanner (Axon Inc.)
Scan protocol
Slides were scanned on a GenePix 4000B laser Scanner (Axon Inc.) using GenePix Pro V6.0/1. Both lasers were set to 100% and the PMT was set to 500 for cy3 and 450 for cy5
Description
tissue from mouse brain with high grade glioma.
Data processing
Images were analyzed using Imagene 6.0 (BioDiscovery Inc.). The output of Image analysis was further processed by in-house developed programs. The log2 ratios of the test/control (values are not background subtracted) were normalized using a subgrid loess, with all flagged spots and clones on the sex chromosome are given a weight of zero. The mean of the replicate measures was taken and the final normalized log2 ratio was plotted against its genomic location.
