Arabsolghar Rita, Rasti Mozhgan
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Iran J Med Sci. 2012 Sep;37(3):187-93.
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.
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.
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.
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.
电穿孔是一种将小干扰RNA（siRNA）导入细胞的重要工具，因为它能有效地转化多种细胞类型。由于每种细胞类型的电穿孔条件都必须通过实验确定，本研究提出了一种优化的电穿孔策略，以可重复且高效地用siRNA转染MDA-MB 468人乳腺癌细胞。
为确定最佳条件，首先在无siRNA的情况下对细胞进行电穿孔，并通过台盼蓝和MTT法测定细胞活力。然后在最佳条件下进行siRNA转染。采用蛋白质免疫印迹分析监测siRNA转染是否成功。
使用Gene Pulser X细胞电穿孔系统，该细胞系电穿孔的最佳条件是指数衰减模式下220伏和975微法。我们的数据表明，通过使用合适的电穿孔条件，与阴性对照siRNA电穿孔相比，10纳摩尔DNMT1 siRNA可使MDA-MB 468细胞中的DNA甲基转移酶mRNA沉默。细胞活力分析表明，最佳电穿孔条件下，通过台盼蓝染色和MTT法测定的细胞活力分别为74%和78%。
在所获得的电穿孔条件下，用siRNA转染MDA-MB-468乳腺癌细胞系成功，实现了有效的基因沉默且细胞活力较高。
