sex: Male age: 75 years chemo-treated: Yes histological differentiation: Poorly days to death or last seen alive: 227
Extracted molecule
genomic DNA
Extraction protocol
On average, 10 30µm cryostat sections were prepared for the extraction of genomic DNA from tumor samples embedded in optimal cutting temperature mounting medium. DNA extracted was then cleaned using phenol:chloroform:isoamyl alcohol and precipitated using ethanol and sodium acetate.
Label
Cy3
Label protocol
DNA labeling was performed using the BioPrime DNA labeling kit reagents (Invitrogen, Carlsbad, CA, USA) and according to protocols described previously (van den Ijssel, P et. al, 2005). 300ng of genomic DNA was mixed with 20µl of 2.5x random primer and miliQ water to a total volume of 42 µl. After the mixture was denatured, Cy3-dCTP (test sample) or Cy5-dCTP (pooled reference sample) and exo-klenow DNA polymerase were added to the mixture and incubated for 16 hours at 37°C. After removing unincorporated Cy3 and Cy5 using ProbeQuant G-50 Micro Spin Columns (GE Healthcare, Little Chalfont, UK), labeled DNA was collected in microfuge tubes. 5 dye-swap experiments were undertaken whereby the test sample was labeled with Cy5 and the reference sample with Cy3 to compare the consistency of data derived.
On average, 10 30µm cryostat sections were prepared for the extraction of genomic DNA from tumor samples embedded in optimal cutting temperature mounting medium. DNA extracted was then cleaned using phenol:chloroform:isoamyl alcohol and precipitated using ethanol and sodium acetate.
Label
Cy5
Label protocol
DNA labeling was performed using the BioPrime DNA labeling kit reagents (Invitrogen, Carlsbad, CA, USA) and according to protocols described previously (van den Ijssel, P et. al, 2005). 300ng of genomic DNA was mixed with 20µl of 2.5x random primer and miliQ water to a total volume of 42 µl. After the mixture was denatured, Cy3-dCTP (test sample) or Cy5-dCTP (pooled reference sample) and exo-klenow DNA polymerase were added to the mixture and incubated for 16 hours at 37°C. After removing unincorporated Cy3 and Cy5 using ProbeQuant G-50 Micro Spin Columns (GE Healthcare, Little Chalfont, UK), labeled DNA was collected in microfuge tubes. 5 dye-swap experiments were undertaken whereby the test sample was labeled with Cy5 and the reference sample with Cy3 to compare the consistency of data derived.
Hybridization protocol
Pre-hybridization mixture was prepared using 50µl of Cy3-labeled test DNA, 50µl of Cy5-labeled reference DNA, 10mg Human Cot-1 DNA (Invitrogen, Carlsbad, CA, USA) and precipitated using 0.1 volume of 3 M sodium acetate pH 5.2 and 2.5 volume of ice-cold 100% ethanol, followed by centrifugation for 30 minutes at 14000 rpm and 4°C. The pellet was re-dissolved in 13 ml of yeast tRNA (100 mg/ml, Invitrogen, Carlsbad, CA, USA) and 26 ml of 20% SDS. 105µl of hybridization solution (4.3% (w/v) dextran sulphate (GE Healthcare, Little Chalfont, UK), 50% (v/v) ultra pure de-ionised formamide (Invitrogen, Carlsbad, CA, USA), 2.9x saline-sodium citrate (SSC) pH 7.0 (Sigma Aldrich, Paisley, UK)) was added and gently mixed. The hybridization mixture was then denatured (73°C, 10 minutes) and incubated (37°C, 60 minutes). Slide hybridization and washing were undertaken using a GeneTAC/HybArray12 hybridization station (Genomic Solutions, Huntingdon, UK), performed for 38 hours at 37°C using a continuous agitation mode. After hybridization, slides were washed for 6 cycles (flow for 10 seconds, hold for 20 seconds) with 50% (v/v) formamide/2x SSC (VWR International, Lutterworth, UK), 2 cycles with phosphate-buffer (0.1 M Na2HPO4, pH 8.0, 0.1% (v/v) Igepal CA630 (Sigma Aldrich, Paisley, UK)), 2 cycles with 0.2x SSC and 2 cycles with 0.1x SSC. Slides were then further washed with 0.01x SSC for 5 minutes whilst rocking and dried by centrifugation (1000 g, 3 minutes).
Scan protocol
The slides were scanned using an Agilent G2505B Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA). Laser power was set at 100%, PMT (photo multiplier tube) set at 100% and the split and rotate TIFF mode selected. Red and green channel TIFF format images were collected.
Description
sample id: D138
Data processing
Spot detection, intensity assessment analysis and quality control were fully automated using BlueFuse version 3.4 Build 5836 (BlueGnome, Cambridge, UK). Spots were excluded if the quality flag was <1 or the confidence level <0.1. Oligonucleotides from the human library were mapped to the human genome build NCBI35. Oligonucleotide sequences and mapping are available from Compugen, San Jose, CA, USA. The raw data were analyzed using snapCGH package of Bioconductor (http://www.bioconductor.org/) and re-scaled using global median normalization to account for the differences in individual hybridization intensities. MA plots were constructed to show the relationship between A (the “average signal” [0.5 x (log R + log G)], where R is the background subtracted red signal, G the background subtracted green and M the log2 R/G ratio. Each plot was reviewed by 2 independent reviewers (Rees JR and Chin SF). A cut-off of 2 times the standard deviation (SD, 0.29) above/below the mean log2 ratio (0.03) was employed for genomic gains/losses covering at least 5 oligonucleotides. A cut off of 3 SD from the mean was set for detection of high-level gains or amplifications and homozygous deletions (HDs). Spot detection, intensity assessment analysis and quality control were fully automated using BlueFuse version 3.4 Build 5836 (BlueGnome, Cambridge, UK). Spots were excluded if the quality flag was <1 or the confidence level <0.1. Oligonucleotides from the human library were mapped to the human genome build NCBI35. Oligonucleotide sequences and mapping are available from Compugen, San Jose, CA, USA. The raw data were analyzed using snapCGH package of Bioconductor (http://www.bioconductor.org/) and re-scaled using global median normalization to account for the differences in individual hybridization intensities. MA plots were constructed to show the relationship between A (the “average signal” [0.5 x (log R + log G)], where R is the background subtracted red signal, G the background subtracted green and M the log2 R/G ratio. Each plot was reviewed by 2 independent reviewers (Rees JR and Chin SF). A cut-off of 2 times the standard deviation (SD, 0.29) above/below the mean log2 ratio (0.03) was employed for genomic gains/losses covering at least 5 oligonucleotides. A cut off of 3 SD from the mean was set for detection of high-level gains or amplifications and homozygous deletions (HDs).
