Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA)
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted after harvesting the cells by Trypsin (Invitrogen) followed by phenol-chloroform extraction and subsequent precipitation in 100% ethanol. DNA precipitate was washed with 70% ethanol then eluted in DNAse free water.
Label
CY3
Label protocol
Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, DNA was labelled with 1.5 - 3 mM Cy5-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).
Comercial DNA extracted as per manifacturers instructions - Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA)
Label
CY5
Label protocol
Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, DNA was labelled with 1.5 - 3 mM Cy3-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).
Hybridization protocol
Probe mixture of Cy3-labelled sample DNA, Cy5-labelled reference DNA, 50 μl of 1.0mg/ ml of human Cot-1 DNA (Invitrogen, USA), 52 μl of Agilent 10X Blocking Agent and 260 μl of Agilent 2X Hybridization Buffer (Agilent Technologies, Inc.) was denatured at 100C for 1 minute 30 seconds and incubated at 37C for 30 minutes. The probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40 hours at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm. Arrays were washed according to the manufacturer’s recommendation and air dried.
Scan protocol
Arrays were scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc).
Description
The Agilent Human Genome CGH microarray 244A (Agilent Technologies, Inc., Palo Alto, USA) were used for the array-CGH experiments. Three μg of Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA) and 3 μg of test genomic DNA sample were digested with AluI (5 units) and RsaI (5 units) (Promega) for a minimum of 2 hours at 37C. Digestion quality was assessed by the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer, Agilent Technologies). Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, the reference and sample DNA were labelled with 1.5 - 3 mM Cy3-dUTP or Cy5-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA). Probe mixture of Cy3-labelled sample DNA, Cy5-labelled reference DNA, 50 μl of 1.0mg/ ml of human Cot-1 DNA (Invitrogen, USA), 52 μl of Agilent 10X Blocking Agent and 260 μl of Agilent 2X Hybridization Buffer (Agilent Technologies, Inc.) was denatured at 100C for 1 minute 30 seconds and incubated at 37C for 30 minutes. The probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40 hours at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm. Arrays were washed according to the manufacturer’s recommendations; air dried, and scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc), and processed using Agilent’s Feature Extraction software. Dye-swapped duplicate experiments were carried out.
Data processing
Data were processed using Agilent’s Feature Extraction software protocol for array-CGH data. To analyze genomic imbalance tumour samples (2 arrays each) the processed R signal and processed G signal columns from the Agilent Feature Extraction-generated a-CGH .txt files were imported into PGS. The tumour-specific signal across all probes was normalized as a ratio to baseline using Normalise to Baseline Tool in PGS, where baseline data corresponded to the normal human DNA. The data was then log2 transformed using the PGS Normalization and Scaling Tool. In order to detect regions of genomic gain and loss we applied the Genomic Segmentation tool with segmentation parameters set at: min. probes: 10, p-value threshold: 0.01, and signal to noise: 0.1. Region report was set at values bellow -.5/+.5 (log2) and p-value threshold of 0.05 for 2/2 (replicate) arrays in individual analysis or at 40% cut-off in cumulative analysis (i.e. 4/10 tumour samples). Regions of significant gain or loss were annotated to the corresponding genes present on the Affymetrix Gene 1.0 Array using the HuGene-1_0-st-v1.na24.hg18.transcript.csv file. Visualizations and VENN analysis were performed in PGS.
