siRNA in Drosophila S2 cells? - (Jul/04/2005 )

Hi there! My first post at this forum and I hope somebody can help me. For my thesis I'm looking at RNAi in Drosophila S2 cells. The silencing with dsRNA works well, but I don't have experience with siRNA. Could somebody tell me what transfection reagent to use and maybe the concentration of siRNA needed?
And is it possible to transfect my plasmid with the GFP-gene to be silenced and the siRNA at the same time?
Well, that's it. Thank you!

I do not have experience with S2 cells. siRNA transfection should be easier than dsRNA (you mean hairpin RNA, right?). siRNA working concentration can be at the range of 5 nM to 50 nM. You can look into the literature for siRNA concentration and duration, and I believe many people have transfected S2 cells.

Yes, you can transfect plasmid and siRNA at the same time, but it is very hard to control the transfection efficiency (they compete for the transfection reagent). Or you can try transfecting the plasmid first, and then splitting the cells for siRNA transfection.

Thank you. Well, for silencing with dsRNA, I just add the dsRNA (no hairpin) to the medium..piece of cake. But I will look for some protocols and try them. Thanks for your answer!

Yes. It is possible to transfect both plasmid and siRNA together into S2.
The key is the molar ratio of these two nucleic acids. My experience is at least 10:1 ratio (siRNA:plasmid) to be able to see the knockdown. Our lab has high success rate using Gencarrier-1 (Epoch biolabs) for co-transfection on S2 cells (with siRNA and a expression plasmid). Good luck.