Optimized basic conditions are essential for successful siRNA transfection into primary endothelial cells.
RNA interference (RNAi) is a powerful technique in basic research and has a high potential for therapeutic applications. To realize its clinical applicability, introduction of short double-stranded RNA (dsRNA) has to be carried out under physiological conditions. This study evaluates two cationic liposomal transfection reagents on the efficiency of successful silencing of primary human endothelial cells. Transfection efficiency was investigated under different conditions, for example different media during transfection, duration of transfection, siRNA concentration, and the use of serum and antibiotics. Viability after transfection was examined by CASY and MTT assay. Interferon response was examined by real-time PCR. First we revealed that transfection carried out in the presence of serum and antibiotics caused good knockdown results only by the use of the novel lipid cationic transfection reagent. Both lipid cations had slightly the same transfection efficiency over the range of 10-150 nM siRNA concentration. Examination of interferon response showed increasing OAS1 and STAT1 expression, but not as high as if the transfections were carried out with synthetic polyinosinic-polycytidylic acid double-stranded RNA (poly[IC]). The optimized combination of basic conditions for transfection significantly enhanced the efficiency of the siRNA-mediated knockdown, without causing toxicity or stimulation of the interferon pathway.
Department of Thoracic, Cardiac, and Vascular Surgery, Division of Sports Medicine, University Hospital Tübingen, Tübingen, Germany.
Andrea
A
Nolte
Claudia
C
Raabe
Tobias
T
Walker
Perikles
P
Simon
Gerhard
G
Ziemer
Hans Peter
HP
Wendel
D002478:Cells, Cultured; D019040:E-Selectin / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007372:Interferons / Q000187:drug effects / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology; D008081:Liposomes; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D019040:E-Selectin; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D034741:RNA, Small Interfering; D007372:Interferons