Gene expression analysis of primary human myeloid precursor cells treated with O6-benzylguanine (6BG) and Temozolomide (TMZ). Homo sapiens

For the comparison of 6BG/TMZ with control sample, the predominant annotation among the upregulated genes is ‘apoptosis’. The majority of the downregulated genes is assigned to heat shock proteins and proteins which bind unfolded proteins. Overall design: To look at early changes in gene expression, primary human myeloid precursor cells (40 x 10^6 per sample) derived from 3 pooled CD34+ products were treated for 18 hours with control (vehicle), 6BG, TMZ, or 6BG/TMZ and cell pellets flash frozen. Total RNA were isolated at Miltenyi Biotec (Cologne, Germany) and bioinformatics analysis of four microarray datasets was performed by their Bioinformatics Group. The direct comparisons were: 6BG/TMZ vs Control, TMZ vs 6BG, 6BG/TMZ vs 6BG, 6BG/TMZ vs TMZ. A two-dye competitive hybridization of mRNAs derived from differently treated human cells in comparison to a reference mRNA derived from cells which underwent a different treatment was conducted. After treatment with two different drugs or a combination of both drugs, respectively, RNA was extracted from the cells and hybridized against the corresponding reference mRNA. As microarray platform, the PIQOR™ Cell Death Microarray with 494 probes was used.

在6BG/TMZ与对照样本的比较分析中，上调基因的主要注释类别为细胞凋亡（apoptosis）。大部分下调基因则被注释为热休克蛋白以及未折叠蛋白结合蛋白。
实验整体设计：为探究基因表达的早期变化，将源自3份混合CD34+产物的原代人髓系前体细胞（每份样本含40×10^6个细胞）分别以对照溶剂、6BG、TMZ或6BG/TMZ处理18小时，随后快速冷冻细胞沉淀。总RNA提取工作在德国科隆的美天旎生物技术有限公司（Miltenyi Biotec）完成，该公司生物信息学团队对4个微阵列数据集开展了生物信息学分析。
直接比较组包括：6BG/TMZ vs 对照组、TMZ vs 6BG、6BG/TMZ vs 6BG、6BG/TMZ vs TMZ。本实验采用双染料竞争性杂交策略：将经不同处理的人源细胞mRNA与经其他处理方式得到的参考mRNA进行竞争性杂交。分别经两种单一药物或两种药物联合处理后，提取细胞总RNA，并与对应的参考mRNA进行杂交。实验所用微阵列平台为搭载494个探针的PIQOR™细胞死亡微阵列。