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

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. Overall design: 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)