Gene expression profiles of oxaliplatin resistant colorectal cancer and ovarian cancer cell lines

Oxaliplatin resistance was induced in 2 colorectal cancer cell lines (LoVo-92, wt-p53 and LoVo-Li, functionally inactive p53) and one ovarian cancer cell line (A2780, wt-p53). Resistance was induced by weekly exposure to oxaliplatin for 4 hrs or 72 hrs with increasing concentrations for a period of 7 months. After RNA isolation, 500ng of RNA of the resistant and parental cell line were linearly amplified and fluorescently labeled with either Cy3-CTP or Cy5-CTP with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Amstelveen, The Netherlands). Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 4x44K Whole Human Genome arrays (Agilent Technologies) according to the manufacturer. Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 44K Whole Human Genome arrays (Agilent Technologies, Part Number G4112A) according to manufacturer's instructions. Microarrays were scanned using an Agilent DNA Microarray Scanner, and scans were quantified using Agilent Feature Extraction software (version 8.5.1). Raw expression data generated by the Feature Extraction software was imported into R using the LIMMA package in Bioconductor (http://www.bioconductor.org). As overall background levels were very low, no background correction was performed. The intensity distributions within and between arrays were normalized using the quantile scaling algorithm in LIMMA. After normalization, the separate intensity channels were extracted from the ratio measurements and combined to the intensities of the reference. The parental cell line was used as reference.

奥沙利铂（Oxaliplatin）耐药模型于2株结直肠癌细胞系（LoVo-92，野生型p53（wt-p53）；LoVo-Li，功能失活型p53）及1株卵巢癌细胞系（A2780，野生型p53）中构建完成。耐药模型的构建方式为：以递增浓度的奥沙利铂每周处理细胞4小时或72小时，持续时长共计7个月。RNA分离完成后，取耐药株与亲本株的RNA各500 ng，采用安捷伦低RNA输入荧光线性扩增试剂盒（Agilent Low RNA Input Fluorescent Linear Amplification Kit，安捷伦科技公司（Agilent Technologies），荷兰阿姆斯特丹）进行线性扩增，并分别以Cy3-CTP或Cy5-CTP完成荧光标记。将等量（1 μg）的Cy3-CTP与Cy5-CTP标记样品按照制造商说明书，与安捷伦4x44K全人类基因组微阵列芯片（Agilent Technologies）进行杂交；后续将等量的上述标记样品按照制造商操作指南，与安捷伦44K全人类基因组微阵列芯片（Agilent Technologies，货号G4112A）进行杂交。使用安捷伦DNA微阵列扫描仪对微阵列芯片进行扫描，并通过安捷伦特征提取软件（版本8.5.1）对扫描结果进行定量分析。通过Bioconductor中的LIMMA软件包，将特征提取软件生成的原始表达数据导入R语言分析环境。鉴于整体背景信号水平极低，未开展背景校正操作。采用LIMMA中的分位数缩放算法，对芯片内及芯片间的信号强度分布进行标准化处理。标准化完成后，从比值测量结果中提取独立的强度通道，并结合得到参考样本的信号强度，本研究以亲本细胞系作为参考样本。