siRNA transfection problem - (Dec/21/2006 )
Hi,guys, currently i'm doing siRNA transfection to Hela cells with Fugene 6. However, after overnight incubation, almost 90% of cells are floating in the medium which are supposed to be dead cells. The concentration of siRNA i'm using for transfection is 35nM which is within the range on the manual provided and much lower than some papers(100nM-150nM). So anybody can help me out with this problem? Thx.
Some basics questions:
1. Do you have a fugene alone control without any si RNA?? How does that look?? If it looks bad then it might be the quality or quantity of fugene being used. Use a different bottle or decrease the quantity of fugene.
2. is the gene that you are knocking out very crucial for growth?? If so then that might be the resaon for your dead cells.
3. How did you determine that 35 nM concentration. Did you do a titration experiemnt??
True. I'd also do an siRNA control, too (maybe just one well).
i've transfected siRNA to 125nM concentration and no problem of such dea cells
1 - what is the protein targeted ?
2 - what is the quantity of Fugene used?
3 - do a FUGENE only treatment and compare to your fugnene + siRNA you will be able to see if it's from your siRNA or from your fugene treatment
if you use too much fugene, hela gonna dead surely
How much fugene are you using? Fugene is the best transfection reagent for plasmids in most cell lines we use (including HeLas) but it can be toxic at higher concentrations if you are transfecting a lot of DNA.
Definitely use a control siRNA to check that your gene isn't essential, but I would also try lipofectamine. Using a FITC-tagged siRNA we get about 10-15 times greater transfection of HeLa's as measured by flurescence on a FACS calibur when using Lipofectamine compared to Fugene!