Global Gene Expression Profiles of Subcutaneous Adipose in Females with Type 2 Diabetes.. Homo sapiens

Gene expression profiles of biopsy samples of subcutaneous adipose of three female patients of type 2 diabetes and three non-diabetic female patients were generated using Illumina HumanHT-12 v3 Expression BeadChip arrays. The primary indications of surgery were non-infective and non-malignant conditions, namely, cholelethiasis, hernia and trauma. Overall design: Three biological replicates with four technical replicates each were used to generate expression profiles. Total cellular RNA from biopsy samples was isolated using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX). The quantity and the quality of RNA samples were determined using Nanodrop-1000 (Thermo Fischer Scientific, Wilmington, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), in that order. RNA samples with a RIN (RNA Integrity Number) value between 5-8 were used for expression profiling. Starting with 500 ng of total RNA, Illumina TotalPrepTM RNA Amplification Kit was used for preparing first and second strand cDNA, purification of cDNA, in vitro transcription to synthesize biotin labeled cRNA, and purification of the labeled cRNA, in that sequence. The quantitation of cRNA was performed using Nanodrop-1000. Illumina HumanHT-12 v3 Expression BeadChip arrays were hybridized with 750 ng of labeled cRNA samples. Hybridization and washing were perforemd according to the manufacturer's protocol. The arrays were scanned and read using Illumina iScan System, and the data extraction, average normalization and downstream analysis performed using Illumina GenomeStudio V2010.1.

本数据集针对3名2型糖尿病女性患者与3名非糖尿病女性患者的皮下脂肪活检样本进行基因表达谱分析，检测平台采用Illumina HumanHT-12 v3表达微珠芯片阵列(Illumina HumanHT-12 v3 Expression BeadChip arrays)。所有受试者的手术主要适应证为非感染性、非恶性疾病，具体包括胆囊结石病、疝与创伤。

研究设计概况：本研究设置3组生物学重复，每组生物学重复包含4次技术重复，以此生成基因表达谱数据。

总RNA提取：采用mirVana™ miRNA分离试剂盒（Ambion，美国得克萨斯州奥斯汀市）从活检样本中分离总细胞RNA。RNA样本的浓度与质量依次通过Nanodrop-1000分光光度计（赛默飞世尔科技，美国特拉华州威尔明顿市）与Agilent 2100生物分析仪（安捷伦科技，美国加利福尼亚州圣克拉拉市）进行评估，仅选取RNA完整性指数(RNA Integrity Number, RIN)介于5~8之间的样本用于后续表达谱分析。

样本扩增与标记：以500 ng总RNA为起始量，按照顺序使用Illumina TotalPrep™ RNA扩增试剂盒依次完成第一、第二链cDNA合成、cDNA纯化、体外转录合成生物素标记的cRNA以及标记cRNA的纯化操作。通过Nanodrop-1000完成cRNA的定量后，取750 ng定量后的标记cRNA与Illumina HumanHT-12 v3表达微珠芯片阵列进行杂交，杂交与洗涤步骤严格遵循制造商的实验方案执行。

数据采集与分析：使用Illumina iScan系统对芯片进行扫描与信号读取，并通过Illumina GenomeStudio V2010.1软件完成数据提取、均值归一化及下游生物信息学分析。