Osteosarcoma Copy Number Analysis. Homo sapiens

Osteosarcoma (OS) is a very aggressive bone tumor characterized by highly abnormal complex karyotypes.This a-CGH is a part of an expriment whose aim was to identify, genomic imbalance, DNA methylation and gene expression profiles in a panel osteosarcoma tumors. Keywords: comparative genomic hybridization Overall design: The Agilent Human Genome CGH microarray 244A (Agilent Technologies, Inc., Palo Alto, USA) were used for U2OS array-CGH experiments. Three μg of Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA) and 3 μg of test genomic tumour 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 Cy5-dUTP or Cy3-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.

骨肉瘤（Osteosarcoma, OS）是一类极具侵袭性的骨肿瘤，以高度异常的复杂核型为特征。本次阵列比较基因组杂交（array-based Comparative Genomic Hybridization, a-CGH）实验旨在通过一组骨肉瘤样本，鉴定基因组失衡、DNA甲基化与基因表达谱特征，本数据集为该实验的一部分。
关键词：比较基因组杂交（Comparative Genomic Hybridization, CGH）

整体实验设计：
本实验采用安捷伦人类基因组CGH微阵列244A（Agilent Human Genome CGH microarray 244A，安捷伦科技公司，美国帕洛阿尔托）开展U2OS细胞系的阵列CGH实验。取3 μg 来自多名匿名男性供体的人类基因组DNA（Promega公司，美国麦迪逊）与3 μg 待测肿瘤基因组DNA样本，使用AluI限制性内切酶（5 U）与RsaI限制性内切酶（5 U，Promega）于37℃酶切至少2小时。采用DNA 1000 LabChip试剂盒（安捷伦2100生物分析仪，安捷伦科技）评估酶切质量。参照试剂盒说明书，使用安捷伦基因组DNA标记试剂盒PLUS（Agilent Genomic DNA Labeling Kit PLUS，安捷伦科技公司，美国帕洛阿尔托）完成标记反应。简言之，参考DNA与待测DNA分别用1.5~3 mM的Cy5-dUTP或Cy3-dUTP（安捷伦科技公司，美国帕洛阿尔托）标记，并通过Centricon YM-30超滤管（默克密理博，美国比勒里卡）纯化。将Cy3标记的待测DNA、Cy5标记的参考DNA、50 μL 1.0 mg/mL人类Cot-1 DNA（Invitrogen，美国）、52 μL 安捷伦10X封闭试剂与260 μL 安捷伦2X杂交缓冲液（安捷伦科技公司）混合制成探针体系，于100℃变性1分30秒，随后在37℃温育30分钟。使用安捷伦微阵列杂交腔将探针加至芯片，于65℃、20 rpm旋转杂交炉（Robbins Scientific，美国森尼韦尔）中杂交40小时。按照试剂盒说明书洗涤芯片，空气干燥后使用安捷伦2565AA DNA微阵列扫描仪（安捷伦科技公司）扫描，并通过安捷伦特征提取软件完成数据分析。同时开展了染料互换的重复实验。