Streptolysin-O reversible permeabilisation is an effective method to transfect siRNAs into myeloma cells
- 作者：Jose L.R. Brito ; Faith E. Davies ; David Gonzalez ; Gareth J. Morgan
- 关键词：Multiple myeloma ; Streptolysin-O ; siRNA ; Transfection
- 刊名：Journal of Immunological Methods
- 出版年：2008
- 期刊代码：57_00221759
- 类别：imm
- 出版时间：20 April 2008
- 卷：333
- 期：1-2
- 页码：147-155
- 文件大小：814 K
摘要
RNA interference (RNAi) has been shown to be a valuable tool to specifically target gene expression in a number of organisms becoming an indispensable weapon in the arsenal in functional genomics. In this study, we demonstrate that streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. We used published, pre-validated siRNAs for ERK2 and non-silencing siRNA control. We transfected siRNAs into human myeloma cell lines using SLO reversible permeabilisation method. Flow cytometry and western blot analysis were performed to assess the effect of SLO on transfection efficiency and ERK2 knockdown. These experiments demonstrate that SLO reversible permeabilisation method is an efficient and easy-to-use method to deliver siRNAs into human myeloma cell lines. Optimised SLO permeabilisation method showed to transfect > 80%of JIM-3, H929, RPMI8226 and U266 cells, with minimal effect on cell viability (< 10%) and cell cycle. Equally important, SLO permeabilisation induced a substantial knockdown of ERK2 at the protein level. These studies demonstrate that reversible SLO permeabilisation can successfully be applied to hard-to-transfect human myeloma cell lines to effectively silence genes.