Optimized Basic Conditions Are Essential for Successful siRNA Transfection into Primary Endothelial Cells
| Autor: | Hans Peter Wendel, Andrea Nolte, Gerhard Ziemer, Perikles Simon, Claudia Raabe, Tobias Walker |
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| Rok vydání: | 2009 |
| Předmět: |
animal structures
viruses Biology Transfection RNA interference Interferon Genetics medicine Humans MTT assay RNA Small Interfering Molecular Biology Cells Cultured Liposome Gene knockdown fungi Endothelial Cells RNA Lipids Molecular biology Cell biology RNA silencing Liposomes embryonic structures Molecular Medicine Indicators and Reagents Interferons E-Selectin medicine.drug |
| Zdroj: | Oligonucleotides. 19:141-150 |
| ISSN: | 1557-8526 1545-4576 |
| DOI: | 10.1089/oli.2009.0182 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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