siRNA plus drugs - weird cell morphology (May/02/2008 )
I am working with siRNA to silence gene expression and adding cdk inhibitors to see if I can increase certain aspects of cellular processes. The problem is that when I add the inhibitors with the siRNA complexes (we use lipid based transfection) just a day later the cells begin to appear very strangely. It almost looks like there is contamination (but please trust me its not because this has happened three times). This even occurs with non-specific siRNA. I was wondering if anyone else is working with silencing using siRNA and adding ANY drugs and seeing if the drug inteferes with the processing of the RNAi? I have tried transfecting and adding drug at the same time with drug addition occuring at the very last second. Any suggestions on protocol such as how to add either component to my cells would be greatly appreciated. Thanks.
How did the group with drug treatment alone look like?
That is what is intriguing and very frustrating at the same time. The drug treatment alone killed off the cells (which is what is supposed to happen) but in an orderly way (most likely apoptotically) with no strange morphology. I believe the siRNA is very very sensitive to the slight toxic effects of the drug. I am trying to figure out the best time course of administration for both (drug for 24 hours then transfection for example) but cant seem to find the right combination. The transfection works fine alone so does the drug but cant figure it out. Any suggestions would be greatly appreciated. thanks
The best I can say is that transfection process with this reagent is not harmless to cells and may activate some signaling pathway and sensitize the drugs action, as indicated in your control siRNA group. I would suggest using different transfection protocol, either reduce the lipid to siRNA ratio and/or total complex dosages for the control siRNA group with drug treatment, until you feel you can overcome this problem. If it still does not work, you may have to switch to different reagent, by electroporation or even viral infection method to introduce RNAi.