Genomic DNA was isolated from peripheral blood lymphocytes with the PureGene DNA Purification Kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
Genomic DNA was completely fragmented with AluI and RsaI (Promega, Madison, WI, USA ), followed by purification with QIAprep Spin Miniprep Kit (Qiagen). All of the digested DNAs were subjected to labeling reactions using Agilent’s Genomic DNA Labeling Kit PLUS per manufacturer’s instructions.
reference: commercialized normal Caucasian samples
Extracted molecule
genomic DNA
Extraction protocol
not applicable, commercialized normal Caucasian samples purchased from Promega, Madison, WI, USA.
Label
Cy3
Label protocol
Genomic DNA was completely fragmented with AluI and RsaI (Promega, Madison, WI, USA ), followed by purification with QIAprep Spin Miniprep Kit (Qiagen). All of the digested DNAs were subjected to labeling reactions using Agilent’s Genomic DNA Labeling Kit PLUS per manufacturer’s instructions.
Hybridization protocol
Array hybridizations were carried out with 490μl of hybridization mixtures for 40 h at 65℃, then washed for 5 min at room temperature in Oligo aCGH Wash Buffer 1 (Agilent), followed by 1 min at 37℃ in Oligo aCGH Wash Buffer 2 (Agilent).
Scan protocol
The hybridized arrays were scanned using an Agilent G2565BA DNA microarray scanner (Agilent Technologies). The ScanControl Software (Agilent Technologies) was used for image acquisition. In brief, the arrays were imaged at a resolution of 5 μm. Excitation of Cy3 and Cy5 was performed at a wavelength of 532 and 635 nm, respectively. A laser power of 100% was used. Each array was subjected to a series of scans. The signals were digitized into 16 bit/pixel, yielding a maximum detection range from 1 to 65536; i.e., almost five orders of magnitude.
Description
G4410B
Data processing
After image acquisition, data extraction, normalization and transformation were carried out by using Agilent Feature Extraction Software, version 8.1.1 (Agilent Technologies), procedures listed as follow: (1) Place Grid, find and measure spots -define what is the array format of the image to be processed. (2) Flag outliers a. Compute population outliers- flag features and local backgrounds as population outliers if the minimum number of replicates is met and the mean signals varies significantly from the distribution. b. Compute NonUniform outliers- flag features and local backgrounds as NonUniform if the pixel distribution is outside the boundary defined by the three-parameter noise model. (3) Compute Background, bias and error Background subtraction→ signal correction → choose error model (4) Dye normalization – a. Use rank consistent probes. b. Normalization correction methods: Linear method (5) Compute ratios- the log ratio values are capped to 4 with appropriate signs in positive and negative directions. Subsequent data analysis was performed with the other custom analytical software, CGH Analytics (version 3.5.14, Agilent Technologies). The Linear smoothing algorithm is used in the Moving Average approach to smooth the data sets with window size of 5 Mb.
