Oligo acgh chip on chip hybridization kit

The genomes of haploid wild-type, cell-fusion, and mutant strains were analyzed using a tiling array (Agilent Technologies, Santa Clara, CA, USA). Genomic DNA was extracted using spin columns (Genomic DNA Buffer Set No. 19060, and Genomic tip 500/G No. 10262; Qiagen, Hilden, Germany). A. thaliana genomic DNA was extracted using spin columns (DNeasy Plant Mini Kit No. 69104; Qiagen). For tiling-array analysis, array comparative genomic hybridization (aCGH) was performed using a S. cerevisiae or A. thaliana custom microarray (designed by eArray; Agilent Technologies) for genome-wide copy number analysis. Genomic DNA (100 ng) was fluorescently labeled using the SureTag DNA Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Prior to hybridization, sample and control probes were incubated with the blocking agent and hybridization buffer contained in the Oligo aCGH/ChIP-on-chip Hybridization Kit (Agilent Technologies) at 95 °C for 3 min followed by 37 °C for 3 min. Hybridization was performed at 65 °C for 24 h in a microarray hybridization oven (Agilent Technologies) according to the manufacturer instructions, and the slides were washed with the Oligo aCGH/ChIP Wash Buffer Kit (Agilent Technologies). Array data were quantified and normalized using Feature Extraction Software (v.11.0.1.1; Agilent Technologies).

Arrays used in the present study were designed to cover genomic regions bearing the cancer-associated genes by SurePrint G3 Cancer CGH+ single nucleotide polymorphism (SNP) Microarray Kit, 4 × 180 K (Agilent, Santa Clara, CA, USA). Instead of commercial DNA included within the kit, DNA isolated from the adjacent tissue from each patient was used as a source of reference DNA. For DNA labeling with Cy5 and Cy3 labels, Sure Tag Complete DNA Labeling enzyme kit was purchased (Agilent, Santa Clara, CA, USA); Cy5 label was applied as a reference for adjacent tissues and Cy3 label for adenoma tissues. The input amount of DNA into the labeling was on average 850 ng. Hybridization was performed by using an Oligo aCGH/ChIP-on-chip Hybridization kit (Agilent, Santa Clara, CA, USA).

Array-based CGH was performed by the manufacturer Takara Bio Dragon Genomics Center (Seta, Shiga, Japan). In brief, DNA (2 micro g) was fluorescent-labeled by random priming DNA synthesis in the presence of Cy3-dUTP (control group) or Cy5-dUTP (patient group) (Genomic DNA Enzymatic Labeling Kit; Agilent Technologies, Hachioji, Tokyo, Japan). DNA labeling efficiency was estimated by spectrophotometry (Nanodrop ND-2000®) measuring optical absorbance at 260 nm for DNA, at 550 nm for Cy5, and at 649 nm for Cy3. Cy5- and Cy3-labeled DNAs were randomly paired, mixed, and hybridized to SurePrint G3 Human CGH Microarrays (1 M) in the presence of human Cot-1 DNA (Oligo aCGH/ChIP-on-chip Hybridization Kit, Agilent Technologies). Following hybridization for 24 h, microarray slides were washed according to the manufacturer’s instructions and immediately scanned on a DNA Microarray Scanner (Agilent Technologies). With the given limitation of the sample number, we took an advantage of the above direct comparison between case and control samples [57 (link)]. This approach allowed us to determine relative ratios of their gene dosages but not their absolute gene dosages. However this procedure decreased data deviations, compared with the CGH analysis utilizing two microarrays and reference genome DNA [30 (link)].