Sample Extraction Protocol Other: Genomic DNA was extracted from a short-term cultured tumor cells or PBMC using Qiagen Mini genomic kit (Qiagen, Valencia, CA). PROTEINASE K (Qiagen)was added to a solution containing 5x106 cells,PBS and AL buffer and mixed thoroughly by pipetting. The homogenate was then incubated at 56C for 10 minutes and centrifuged for 1 min at 13000 rpm at room temp. The mixture (approximately 400 microl) was then combined and mixed well with one volume of ethanol (96-100%) and transferred into a QIamp spin column placed in a 2ml collection tube. Genomic Dna was than isolated according to the manufacturer protocol.
Label
cy3
Label protocol
Cy3 Labeling Protocol Other: Four μg of genomic DNA were digested for 2 hours with Dpn II (New England Biolabs, Beverly, MA), followed by phenol-chloroform extraction. the labeling reaction was performed according to ARRAY CGH GENOMIC LABELING PROTOCOL (Invitrogen Cat# 18095-011. Digested DNA was mixed with 15 μg of random octamers (provided by kit) in a total volume of 41 μl reaction buffer and heated at 95ºC for 5 min. After chilling on ice, 5μl of 10x-dUTP Nucleotide Mix (provided by Kit, Cy3-dUTP (Amersham, Piscataway, NJ), and 1 μl of Exo-Klenow were added. The labeling reaction was completed at 37 ºC for 2 hours. Reactions were terminated by adding 5 μl of 0.5 M EDTA, pH 8.0. Target clean up: Add 45 µl of TE (pH= 8.0) to each tube Add 400 µl of purification buffer A (provided by kit) to each tube and vortex for 30 seconds Place the purification column(provided by kit) in a 2-ml collection tube, and load the sample into the column. Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through, place the column back in the tube. Add 600 µl of purification buffer B (provided by kit). Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through, place the column back in the tube Add 200 µl of purification buffer B (provided by kit) prepared to the column. Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through. Place the purification column in a new 1.5 ml collection tube Add 50 µl of sterile water and incubate at room temperature for 1 minute. Centrifuge at 11 000 xg for 1 minute at room temperature. The flow-through contains your purified labeled DNA probes.
Sample Extraction Protocol Other: Genomic DNA was extracted from a short-term cultured tumor cells or PBMC using Qiagen Mini genomic kit (Qiagen, Valencia, CA). PROTEINASE K (Qiagen)was added to a solution containing 5x106 cells,PBS and AL buffer and mixed thoroughly by pipetting. The homogenate was then incubated at 56C for 10 minutes and centrifuged for 1 min at 13000 rpm at room temp. The mixture (approximately 400 microl) was then combined and mixed well with one volume of ethanol (96-100%) and transferred into a QIamp spin column placed in a 2ml collection tube. Genomic Dna was than isolated according to the manufacturer protocol.
Label
cy5
Label protocol
Cy5 Labeling Protocol Other: Four μg of genomic DNA were digested for 2 hours with Dpn II (New England Biolabs, Beverly, MA), followed by phenol-chloroform extraction. the labeling reaction was performed according to ARRAY CGH GENOMIC LABELING PROTOCOL (Invitrogen Cat# 18095-011. Digested DNA was mixed with 15 μg of random octamers (provided by kit) in a total volume of 41 μl reaction buffer and heated at 95ºC for 5 min. After chilling on ice, 5μl of 10x-dUTP Nucleotide Mix (provided by Kit, Cy5-dUTP(Amersham, Piscataway, NJ), and 1 μl of Exo-Klenow were added. The labeling reaction was completed at 37 ºC for 2 hours. Reactions were terminated by adding 5 μl of 0.5 M EDTA, pH 8.0. Target clean up: Add 45 µl of TE (pH= 8.0) to each tube Add 400 µl of purification buffer A (provided by kit) to each tube and vortex for 30 seconds Place the purification column(provided by kit) in a 2-ml collection tube, and load the sample into the column. Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through, place the column back in the tube. Add 600 µl of purification buffer B (provided by kit). Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through, place the column back in the tube Add 200 µl of purification buffer B (provided by kit) prepared to the column. Centrifuge at 11 000 xg for 1 minute at room temperature, discard the flow-through. Place the purification column in a new 1.5 ml collection tube Add 50 µl of sterile water and incubate at room temperature for 1 minute. Centrifuge at 11 000 xg for 1 minute at room temperature. The flow-through contains your purified labeled DNA probes.
Hybridization protocol
Sample Hybridization Protocol Wash procedure: Washing: 1. 2x SSC, 0.03% SDS at 65C for 5 minutes; 2. Wash with 2x SSC for 5 min at room temperature. 3 Wash with 1x SSC for 5 min at room temperature 4. 0.2x SSC at room temperature for 5 minutes 5. Centrifuge slide at 80-100 g for 3 min to dry. Other: Combine Cy3 and Cy5 Add 400µl of TE (pH=8.0) to each probe Apply labeled probe to Microcon YM 30 Spin 12000 xg for 11 minutes (make volume less than 20 µl) Discard flow-through, wash with 500 µl TE (pH=8.0) and spin 12000 xg for 11 minutes. Add Hybridization buffer mixing:Human Cot1 50 µl (1mg/ml)(Invitrogen, Cat# 15279-011);Yeast t-RNA 25 µl(4µg/µl)(4mg/ml Sigma- Aldrich Cat# R8759); Poly dT-dA 2.5µl (8 µg/µl)(8mg/ml Pharmacia Cat# 27-7988-01);TE (pH=8.0)450µl Mix well add to the filter Spin 12000 xg for 12 minutes Invert the filter to a new tube, spin for 2 minutes, Adjust volume by water up to 32 µl or dry samples if volume more than 32 microl; Make:Probe 32 µl,20 x SSC 6.8 µl,10% SDS 1.2 µl.Mix to minimize bubbles Denature by heating 99ºC for 3-4 minutes Incubate at 37ºC for 30 minutes Pre warm Hyb Chamber at 65 ºC Spin DNA probe for 5 minutes at max rcf Apply target mixture to array slide, add coverslip, place in humidified hyb chamber.Hybridization were conducted at 65oC for 16-18 hours.
BRB ArrayTool Data Processing Calculation Method: Fluorescence intensities generated by Cy5 and Cy3 probes hybridized onto the microarray slides were scanned at 10-ìm resolution on a GenePix 4000 scanner (Axon Instruments, Union City,CA) at variable PMT voltage to obtain maximal signal intensities with < 1% probe saturation. The fluorescence intensity and ratio data for Cy5 and Cy3 were initially analyzed using GenePix 4.3 software. Raw data were retrieved in BRB-ArrayTools format for analysis and normalization(http://linus.nci.nih.gov/BRBArrayTools.html).. Following filter criteria was applied to the data set: 1. spots were excluded if intensity < 100, and spot size less than 25µm 2. genes were filtered out if >50% of experiment with missing data values. 3. log2 transformed data were normalized using lowess smoother, and intensity ratios were truncated if greater than 64 Further data preprocessing was done using webtool preP (http://prep.bioinfo.cnio.es/). Missing data were imputed using KNN imputation method with K=15. Gene location was extracted according to UniGene Cluster IDs from Ensemble and UCSC using IDconverter (http://idconverter.bioinfor.cnio.es/).The pre-processed aCGH data was then segmented using the circular binary segmentation method implemented in ADaCGH (http://adacgh.bioinfo.cnio.es/) to detect regions with abnormal DNA copy number. The smooth parameters were set as defaults.
