This study was carried out in accordance with the principles of the Declaration of Helsinki, and approved by The Samsung Medical Center (Seoul, Korea) Institutional Review Board (IRB) (no. 2010-04-004). A participant in this study gave written informed consent for research and publication of the results. Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and within the protocols approved by the appropriate IRB at the Samsung Medical Center.
Extracted molecule
genomic DNA
Extraction protocol
gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). For the quality control, DNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), and by 1% agarose gel electrophoresis.
Label
Cy5
Label protocol
DNA labeling and hybridization were performed by using the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (V 7.3, 2014). Briefly, 500ng of genomic DNA was digested with Alu1 and Rsa1 restriction enzymes at 37°C for two hours and then random primers were added. Test and Reference DNA samples were then labeled with Cy5-dUTP and Cy3-dUTP respectively, by incubation with Exo-Klenow at 37°C for a further two hours. Excess nucleotides were removed using Agilent Purification Columns. After purification, the labeled DNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA) that measuring A260 (for DNA), A550 (for Cy5) and A649 (for Cy3) was used for determination of DNA and fluorophore concentrations.
This study was carried out in accordance with the principles of the Declaration of Helsinki, and approved by The Samsung Medical Center (Seoul, Korea) Institutional Review Board (IRB) (no. 2010-04-004). A participant in this study gave written informed consent for research and publication of the results. Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and within the protocols approved by the appropriate IRB at the Samsung Medical Center.
Extracted molecule
genomic DNA
Extraction protocol
gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). For the quality control, DNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), and by 1% agarose gel electrophoresis.
Label
Cy3
Label protocol
DNA labeling and hybridization were performed by using the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (V 7.3, 2014). Briefly, 500ng of genomic DNA was digested with Alu1 and Rsa1 restriction enzymes at 37°C for two hours and then random primers were added. Test and Reference DNA samples were then labeled with Cy5-dUTP and Cy3-dUTP respectively, by incubation with Exo-Klenow at 37°C for a further two hours. Excess nucleotides were removed using Agilent Purification Columns. After purification, the labeled DNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA) that measuring A260 (for DNA), A550 (for Cy5) and A649 (for Cy3) was used for determination of DNA and fluorophore concentrations.
Hybridization protocol
Labeled Test (Cy5) and Reference (Cy3) DNA samples were paired and co-hybridized to the SurePrint G3 Human CGH Microarrays, 4×180K (Agilent®) at 67 °C, 20rpm for 24hrs, then washed at room temperature by using the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (V7.3, 2014). The hybridized array was immediately scanned with an Agilent Microarray Scanner (Agilent Technologies, Inc.)
Scan protocol
CGH data were extracted from scanned images (TIFF files) using Feature Extraction software (v11.0.1.1).
Description
Reference human male DNA labeled with Cy3, purchased from Promega (catalog no. G1471)
Data processing
Extracted signals from the scanned microarray image were translated into log10 ratios of the Cy3(green) labeled normal DNA and Cy5(red) labeled test DNA signals in each of all probes, allowing us to measure DNA copy number changes in the experiments using Agilent Genomic Workbench v7.0.4.0 software. Feature extraction was used to extract foreground signal for background subtraction and to correct dye biases for LOWESS normalization. To reduce biased signals between arrays, data was normalized using the limma package (Ritchie et al., Bioinformatics 2007) implemented in R environment. For the overlapped probe ID in the matrix, corresponding values were averaged.
