7 replies to this topic

[Andras]

Dear All,
I'm using Jetprime to transfect siRNA into cells; since I started to work on a viral protein as well, I thought that instead of stable transfections (which looked good on paper, but do not seem to work), and Lentivirus vectors, I simply double transfect the plasmid into the cells along with the siRNA.
I tried it twice, and I get a significant amount of cell death. Does anyone know why, and what I could do? Less plasmid? Less siRNA?
Thank you.

[Andras]

New development: all the cells that received double transfection died; the controls with plasmid only have started to recover. I've been reading about this, and some people suggested in other topics that endotoxin contamination in plasmid preps might be the problem. (That would explain why the single, plasmid transfected cells died en masse. I still don't understand why all the siRNA cells died, though.)
Any thoughts?

[FishDoctor]

what type of cells are you using?  If your using a qiagen plasmid prep kit to prepare you dna it is going to be fine and endotoxin will be low.  It could be the transfection reagent you using?

[Andras]

Thank you for the answer. The cells are HEK293, and the reagent is Jetprime.
In the meanwhile I figured out the reason: plasmid DNA is apparently toxic to cell in high quantities...
I did a concentration series of double transfections, and it worked: up to 250ng/well cell were OK. They started to die around 300...

[gyma]

Andras, on 11 October 2011 - 06:02 AM, said:

Thank you for the answer. The cells are HEK293, and the reagent is Jetprime.
In the meanwhile I figured out the reason: plasmid DNA is apparently toxic to cell in high quantities...
I did a concentration series of double transfections, and it worked: up to 250ng/well cell were OK. They started to die around 300...

are you sure it is the plasmid toxic to cells? because as you reduce the amount of your plasmid, the amount of your transfection reagent should also be reduced. all kinds of reagents should be toxic and it is just a matter of dose. anyway, it is good that problem solved.

[FishDoctor]

I like the freestyle max reagent for 293 cells, great system and low toxicity.  Very easy to use and very efficient.

[virusfan]

I am not sure if what you claimed is cell line specific.
I have used 1000ng plasmid DNA to transfect my microvascular endothelial cells with jetPrime, but I never have massive cell death. I usually incubate the cells with DNA-jetPrime complex for about 5 hours, then I replenish with fresh medium, though the protocol mentions no need to change medium. Previously, I did observe quite a lot of rounded cells (not entire monolayer, but consider a lot to me) when I left the complex with my cells overnight.

[Andras]

Yes, it's quite peculiar. I'm not sure why it might be so. Maybe the expressed protein kills the cells -after all the plasmid encodes a viral IFN inhibitor.