Expression data from gastric cancer and paried normal tissues

Gastric cancer (GC) is one of the most common cancer worldwide. Specific and reliable molecular markers are limited; it is critical to identify new biomarkers for GC to aid in early diagnosis, treatment strategy, and prognosis evaluation. Microarray technology makes it possible to measure the expression levels of thousands of genes, and identifying meaningful and useful molecular targets from these large data. Total RNA was extracted from 10 pairs of GC tissue and adjacent non-tumor mucosa using TRIzol® reagent (Invitrogen, Carlsbad, CA, US) following the manufacturer's instructions. The RNA integrity number (RIN), detected by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US), was used to determine the RNA integrity. Total RNA meeting the specified quality criteria (RIN ≥ 7.0 and 28S/18S ≥ 0.7) were further purified using an RNase-Free DNase Set (Qiagen, Hilden, Germany) and an RNeasy® Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified total RNA was then used for obtaining biotin-labeled cRNA by applying the GeneChip® 3'IVT Express Kit (Affymetrix, Santa Clara, CA, US), according to the manufacturer's instructions. Then, the GeneChip® Hybridization, Wash, and Stain Kit (Affymetrix, Santa Clara, CA, US) was used for performing the array hybridization and wash, with the use of a GeneChip® Hybridization Oven 645 (Affymetrix, Santa Clara, CA, US) and a GeneChip® Fluidics Station 450 (Affymetrix, Santa Clara, CA, US). All arrays were scanned using a GeneChip® Scanner 3000 (Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with the default settings. Raw data were normalized by the MAS 5.0 algorithm in GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US). The significance analysis of microarray (SAM) was used to identify genes that were differentially expressed between the GC and normal tissues. Genes were considered to be differentially expressed when the Tumor versus Normal signal log ratio values were ≥2 or ≤0.5.

胃癌（Gastric cancer, GC）是全球范围内最常见的恶性肿瘤之一。目前特异性强、可靠性佳的分子标志物（molecular markers）较为匮乏，鉴定新型胃癌生物标志物（biomarkers）以辅助早期诊断、治疗策略制定及预后评估，具有至关重要的临床意义。微阵列技术（microarray technology）可实现对数千个基因表达水平的定量检测，从而从大规模数据中筛选出具有研究价值的有效分子靶点。本研究采用TRIzol®试剂（美国Invitrogen公司，加利福尼亚州卡尔斯巴德），按照制造商说明书从10对胃癌组织及配对癌旁正常黏膜组织中提取总RNA（Total RNA）。通过安捷伦生物分析仪2100（Agilent Technologies, 美国加利福尼亚州圣克拉拉）检测RNA完整性数值（RNA Integrity Number, RIN），以此评估RNA的完整性。对于满足既定质量标准（RIN ≥7.0且28S/18S ≥0.7）的总RNA，采用无RNase脱氧核糖核酸酶试剂盒（RNase-Free DNase Set，Qiagen公司，德国希尔德）及RNeasy®微量试剂盒（RNeasy® Micro Kit，Qiagen公司，德国希尔德），严格依照制造商说明书完成进一步纯化。纯化后的总RNA通过GeneChip® 3'IVT Express试剂盒（GeneChip® 3'IVT Express Kit，美国Affymetrix公司，加利福尼亚州圣克拉拉）合成生物素标记的互补RNA（biotin-labeled cRNA），操作流程遵循制造商说明。随后使用GeneChip®杂交、洗涤与染色试剂盒（GeneChip® Hybridization, Wash, and Stain Kit，美国Affymetrix公司，加利福尼亚州圣克拉拉），结合GeneChip®杂交炉645（GeneChip® Hybridization Oven 645）与GeneChip®流体工作站450（GeneChip® Fluidics Station 450，均为美国Affymetrix公司，加利福尼亚州圣克拉拉产品）完成芯片杂交与洗涤操作。所有芯片均通过GeneChip®扫描仪3000（GeneChip® Scanner 3000）及Command Console Software 3.1软件（均为美国Affymetrix公司，加利福尼亚州圣克拉拉产品），以默认参数完成扫描。原始数据采用GeneSpring Software 11.0软件（美国Agilent Technologies公司，加利福尼亚州圣克拉拉）中的MAS 5.0算法进行归一化处理。采用微阵列显著性分析（Significance Analysis of Microarray, SAM）筛选胃癌组织与正常组织间的差异表达基因，当肿瘤与正常组织的信号对数比值（signal log ratio）≥2或≤0.5时，即判定该基因为差异表达基因。