Document Type : Original Article(s)
Authors
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract
Background: Electroporation is a valuable tool for small interfering RNA (siRNA) delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA.
Methods: To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection.
Results: The best condition for electroporation of this cell line was 220 volt and 975 µF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively.
Conclusion: Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability.
Keywords
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