Gene Expression Profiling of human leukemic B-cells. Two-color Microarray-Based Gene Expression Analysis.

Fresh MEC1 cells (chronic lymphocytic leukemia) (ACC-497, DSMZ) previously untreated and treated in cultures in vitro with glycodendrimers (nanoparticle with maltotriose on the surface). MEC1 cells were centrifuged after incubation with drugs or dendrimers. Agilent’s Low Input Quick Amp Labeling Kit was used to generate fluorescent cRNA (complimentary RNA) with a sample input 100ng of total RNA for two-color processing. The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP. Amplification is typically at least a 100-fold from total RNA to cRNA with the use of this kit. cRNA quantification had been done using NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1. Arrays were placed onto the gaskets and than to the slide chamber in rotisserie in a hybridization oven set. Hybrydization was carried out at 65-Celsius degrees for 17 hours. Arrays were scanning using Agilent Scanner. Scan resolution was set up for 3μM. Microarray gene expression profiling. Intensity of gene expression in MEC1 cells before treatment was compared with the gene expression signals in MEC1 cells from the same cell culture after 4 and 48 hours of treatment. MEC1 cells were treated with PPI-G4-OS-Mal-III dendrimer, PPI-G4-DS-Mal-III, Fludarabina. Sample titles represent; A = Cy3 B = Cy5 R = reference (48h incubation) R4 = reference (4h incubation) 1 = PPI-G4-DS-Mal III (1st cell culture flask) (48h incubation) 2 = PPI-G4-DS-Mal III (2nd cell culture flask)(48h incubation) 3 = PPI-G4-OS-Mal III (1st cell culture flask)(48h incubation) 4 = PPI-G4-OS-Mal III (2nd cell culture flask)(48h incubation) 5 = PPI-G4-DS-Mal III (4h incubation) 6 = PPI-G4-OS-Mal III (4h incubation) 7 = FA (4h incubation) 8 = FA (48h incubation)本数据集的实验对象为既往未接受治疗、经体外糖树状大分子（glycodendrimers，表面修饰麦芽三糖的纳米颗粒）培养处理的新鲜慢性淋巴细胞白血病MEC1细胞（ACC-497，德国微生物菌种保藏中心DSMZ）。将MEC1细胞与药物或树状大分子孵育后，通过离心收集细胞。本实验采用安捷伦（Agilent）低起始量快速扩增标记试剂盒，以100ng总RNA为起始样本进行双色标记，生成荧光标记的互补RNA（cRNA，complementary RNA）。该标记体系依托T7 RNA聚合酶（T7 RNA polymerase），可在扩增靶标核酸的同时，掺入经氰基3（Cy3）或氰基5（Cy5）标记的CTP。使用该试剂盒时，总RNA至cRNA的扩增倍数通常不低于100倍。cRNA的定量检测采用NanoDrop ND-1000紫外-可见分光光度计（版本3.2.1）完成。将基因芯片置于垫片上，随后放入杂交炉的旋转架式样品舱中，杂交实验于65℃条件下进行17小时。芯片扫描采用安捷伦扫描仪完成，扫描分辨率设置为3μm。本实验采用微阵列基因表达谱分析（microarray gene expression profiling）技术，对比MEC1细胞在处理前的基因表达强度，与经相同细胞培养体系分别处理4小时、48小时后的基因表达信号。本次实验所用处理试剂包括PPI-G4-OS-Mal-III树状大分子、PPI-G4-DS-Mal-III树状大分子以及氟达拉滨（Fludarabina）。样本命名规则如下：A代表Cy3通道，B代表Cy5通道；R为48小时孵育的参照样本，R4为4小时孵育的参照样本。具体样本对应关系为：1为PPI-G4-DS-Mal III（第1批细胞培养瓶，48小时孵育），2为PPI-G4-DS-Mal III（第2批细胞培养瓶，48小时孵育），3为PPI-G4-OS-Mal III（第1批细胞培养瓶，48小时孵育），4为PPI-G4-OS-Mal III（第2批细胞培养瓶，48小时孵育），5为PPI-G4-DS-Mal III（4小时孵育），6为PPI-G4-OS-Mal III（4小时孵育），7为FA（4小时孵育），8为FA（48小时孵育）。