BCL6 target genes in a Burkitt's lymphoma cell line with inducible BCL6 expression.. Homo sapiens

In order to determine BCL6 target genes an EBV negative Burkitt's lymphoma cell line, DG75, was stably transfected with a tetracycline transactivator and tight doxycycline responsive expression of GFP was established. The endogenous BCL6 genes of this cell line were disrupted by homologous recombination and a BCL6 cDNA downstream of tetracycline responsive elements (TRE) was inserted to produce Bcl6-/-:tetBCL6-HA cells. Westerns demonstrated doxycycline dependent BCL6 expression.Bcl6-/-:tet. BCL6-HA cells (clone AB7) were either grown without doxycycline (control) or with 1 ug/ml doxycycline for 16, 48 or 96 hours. Total RNA was extracted using RNeasy minipreps (Qiagen) and concentration and quality were checked on the NanoDrop® ND- 1000 spectrophotometer (NanoDrop Technologies, USA) and the RNA Nano 6000 kit (Agilent Technologies) on a 2100 Bioanalyzer (Agilent Technologies). One hundred ng of total RNA was processed with the GeneChip® Eukaryotic Whole Transcript Sense Target Labelling Assay kit (Affymetrix) according to the manufacturer's details. Hybridisation and scanning of GeneChips® was carried out at the CSC/IC Microarray Centre, MRC Clinical Sciences Centre Imperial College London and data analysis by Bioinformatics Support Service, Imperial College London. Briefly, pre- processing of data was performed using GeneSpring GX 10.0.2 software (Agilent Technologies) which applied the "Exon RMA16"¸ algorhithm to the data set. Exon RMA16 performs background correction, quantile normalisation, median polish summarisation and variance stabilisation of 16. In background correction, intensity values of each individual array are corrected for non-specific binding by subtracting the average signal intensity of the area between spots from each probe set. Normalisation is required so multiple chips can be compared to each other. Quantile normalisation adjusts the distribution of probe intensity of each array analysed and so that the distribution of probe intensities for each array in a set of arrays is the same. Probe summarisation refers to the conversion of probe level values (there are approximately 26 probes per gene on each GeneChip®) to a single probe set expression value. Variance stabilisation of 16 refers to the addition of the value 16 to the expression values. By increasing the expression value, the variance of the data set is reduced and the distribution (defined by its mean and its variance) is stabilised. Overall design: A control sample at time (t)=0 was compared to samples at t=16 hours, t=48 hours and t=96 hours following addition of doxycycline.

为鉴定BCL6的靶基因，研究人员将EB病毒（Epstein-Barr Virus, EBV）阴性的伯基特淋巴瘤细胞系DG75稳定转染四环素反式激活因子，并建立了受多西环素严格调控的绿色荧光蛋白（Green Fluorescent Protein, GFP）表达体系。该细胞系的内源性BCL6基因通过同源重组被敲除，并在四环素反应元件（Tetracycline Responsive Element, TRE）下游插入BCL6互补DNA（complementary DNA, cDNA），最终构建得到Bcl6-/-:tetBCL6-HA细胞。蛋白质免疫印迹实验证实BCL6的表达呈多西环素依赖性。将Bcl6-/-:tetBCL6-HA细胞（克隆株AB7）分别置于不含多西环素的培养基（对照组）以及含1 μg/ml多西环素的培养基中培养16、48或96小时。
使用RNeasy迷你试剂盒（Qiagen）提取总RNA，通过NanoDrop® ND-1000分光光度计（美国NanoDrop Technologies公司）以及2100生物分析仪（安捷伦科技）搭配RNA Nano 6000试剂盒检测RNA的浓度与质量。取100 ng总RNA，按照制造商说明书，使用GeneChip®真核生物全转录义链靶标标记试剂盒（Affymetrix）进行处理。
GeneChip®芯片的杂交与扫描实验在伦敦帝国理工学院MRC临床科学中心CSC/IC微阵列中心完成，数据分析由伦敦帝国理工大学生物信息支持服务团队执行。简要而言，数据预处理采用GeneSpring GX 10.0.2软件（安捷伦科技）完成，该软件将"Exon RMA16"算法应用于本数据集。
Exon RMA16算法依次对数据集执行背景校正、分位数归一化、中位数抛光汇总以及16常量方差稳定化处理。在背景校正步骤中，通过从每个探针组的信号强度中减去斑点间区域的平均信号强度，校正各阵列的非特异性结合所带来的强度值偏差。归一化处理可实现多芯片间的相互比较，因此为实验必需步骤。分位数归一化会调整所分析的各阵列的探针强度分布，使得一组阵列中每个阵列的探针强度分布完全一致。探针汇总指将探针水平的数值（每块GeneChip®芯片上每个基因对应约26个探针）转换为单一的探针组表达值。16常量方差稳定化指向表达值添加常数16。通过提升表达值，可降低数据集的方差并稳定由均值与方差共同定义的分布特征。
实验整体设计：以加入多西环素时（t=0）的对照样本，分别与加入多西环素后16小时、48小时及96小时的样本进行比较。