siRNA Transfection - Transfection Protocol
Synthesis of siRNA for RNAi in Drosophila - CSH …
The EGFP stable transfected cell line MDA-MB-231-EGFP cells were used for these studies. Briefly, the cells were grown at 37 °C in humidified atmosphere in L15 medium supplemented with 1% antibiotics, 1% NEAA and 10 % FBS. inhibition on EGFP expression was evaluated by treating the cells with formulations containing PBS, negative control siRNA, naked siRNA, lipofecter/siRNA complex, LMWP/siRNA physical mixture, the bio-reducible Conj-1, or the non-reducible Conj-2). The concentration of siRNA was maintained at 50 nM in all of these formulations. The treated cells were incubated for 12h, followed by the replacement with fresh culture media and further incubation for up to 3 days at 37 °C. The siRNA gene silencing down effect were assessed by the inhibition on EGFP expression, using different methodologies including confocal microscopy, FACS, fluorescence intensity and Western-blot analysis. Experimental details were described in each of the following sections.
Protocol Synthesis of siRNA for RNAi in Drosophila
MDA-MB-231-EGFP cells were seeded at 2×104 cells per well on a glass cover slip for 24 h. After transfection using the aforementioned procedure, cells were rinsed with PBS, fixed with 4% paraformaldehyde solution for 30min at room temperature, and then washed three times with PBS. The media was replaced with 2 mM DAPI (Sigma, USA) Hanks balanced salt solution to stain the nuclei for 10 min, and the cover slip was then placed on a sticky microscopy slide in the presence of the antifade mounting medium (Genemed, USA). The cells were imaged immediately using the Olympus FV-300 laser scanning confocal microscope which was equipped with the FLUOVIEW software (Olympus, Tokyo, Japan).