6 A number of studies have shown that the best way for siRNA transfection is electroporation.7-11 There are pre-set protocols for some cells, but the condition of electroporation for each cell is different. The protocols are pre-programmed with the optimal parameters for some cells. Therefore, the condition of electroporation for other cells must be determined experimentally. In Inhibitors,research,lifescience,medical order for a successful electroporation of siRNA into each cell, optimal

factors for each cell type should be determined. Factors that affect efficient electroporation and viability of cells include waveform, pulse duration, field strength, type and density of cells, and type and concentration of nucleic acid.1,11 There are two types of pulses including square wave and exponential decay. Square wave is used in multiple pulses, and each pulse is applied to the cells for a constant charge and time. The exponential decay pulses rely on an initial voltage, which decays during a constant time. The duration of the decay is controlled by the capacitance setting Inhibitors,research,lifescience,medical and the resistance of the sample, which is constant and affected by the ionic strengths of the electroporation Inhibitors,research,lifescience,medical buffer. When low–resistance buffer (high ionic

strength) such as phosphate buffer saline (PBS) or serum-free growth media are used for mammalian cells, the time-constant is manipulated by selecting the proper capacitance.12 Usually the efficiency of the optimal protocol is confirmed by western blot Inhibitors,research,lifescience,medical analysis.13 In this study, we describe the process of optimizing electroporation condition for siRNA introduction into a human breast cancer cells (MDA-MB-468), which is an estrogen receptor (ER)-negative cell line. For this purpose, we used siRNA to knockdown or downregulate DNA methyl transferase 1 gene (DNMT1) in the MDA-MB-468 cells. To our knowledge, Epigenetics Compound Library chemical structure electroporation-mediated gene silencing Inhibitors,research,lifescience,medical in the MDA-MB-468 cells has not been reported so far. Materials and Methods Cell Culture MDA–MB 468 cell line was obtained from Pasteur Institute, and was cultured in RPMI 1640 with 10% fetal bovine serum and 1% penicillin–streptomycin (37˚C

and 5% CO2). The cells were grown to reach SB-3CT 90% confluence, so they were actively growing on the day of electroporation. Electroporation The cells were washed with PBS, harvested with Trypsin-EDTA, suspended in RPMI media, counted, and diluted with media to a cell density of 5×105 of cells/ml. Typically, 5×105 cells were added to a mixture of 50 µlof serum-free medium and 50 µll sterile distilled water (DW) with or without siRNA. The RNA-cells mixtures were transferred into 4 mm Biorad Gene Pulser cuvettes, and electroporated at different conditions (table 1) by Gene Pulser X cell Electroporation system (BioRad, US). After electreporation, an appropriate amount of the complete medium was added to each aliquot of the cells rapidly and re-plated on to T25 flask or subjected to other tests.