NPC stem-like cells [Agilent]. Homo sapiens

Stem-like cells of nasopharyngeal cancer were enriched by 3 different methods: 1) Side population (SP) assay, 2) LMP2A over-expression and 3) spheroid culture. SP cells and LMP2A over-expressed cells were inoculated into immunocompromised mice for xenograft tumors. Transcriptomes of all 5 of these in vitro and in vivo generated NPC stem-like cells were then analyzed and compared to their counterparts by microarray. All samples were analyzed in the Agilent platform except the SP cells and their counterpart (analyzed in the Affymetrix platform). Overall design: Two to three RNA sample replicates were prepared from each type of the NPC stem-like cells and their counterpart. Dye swap was performed to avoid bias. Each Agilent array was loaded with equal amount of 2 differentially labeled c-RNAs for hybridization. After conversion of scanned microarray images into signal intensities and dye differences corrected in Feature extraction, all analysis was performed in the single color analysis module in GeneSpring.

鼻咽癌干细胞样细胞通过三种不同方法富集获得：1）侧群（Side population, SP）分析法，2）LMP2A过表达处理，3）球体培养法。将SP细胞与LMP2A过表达细胞接种至免疫缺陷小鼠体内以构建移植瘤模型。随后通过微阵列技术，对体外及体内实验获得的共5株鼻咽癌干细胞样细胞的转录组进行分析，并分别与其对应的对照细胞进行比较。除SP细胞及其对照细胞采用Affymetrix平台检测外，其余所有样本均通过安捷伦（Agilent）平台完成分析。
实验设计概况：针对每一类鼻咽癌干细胞样细胞及其对应对照细胞，均制备2~3份RNA样本重复样；通过染料交换实验以规避实验偏差。每块安捷伦（Agilent）芯片均加入等量的两份经差异标记的互补RNA（c-RNA）进行杂交反应。将扫描得到的微阵列图像转换为信号强度值，并通过特征提取（Feature Extraction）软件校正染料差异后，所有分析均在GeneSpring软件的单色分析模块中完成。