performing comparison of different transfection agents - (Aug/08/2005 )

Hi.

I am going to be comparing 4 transfection agents for transfecting siRNA into the U2OS line. I'm just getting started so I was wondering if anyone had any hints for me.

the reagents I'll be comparing are Lipofectamine 2000, HiPerfect, TransIT-TKO, and Fugene 6

I was going to set up an initial transfection to compare using a alexafluor 546 non-specific siRNA to measure efficiency, but was hung up on what conditions to test.

I was planning on a simple initial transfection using all 4 reagents using one cell density in 96 wells, one siRNA concentration, and one reagent amount. would this work? Or should I do the side-by-side comparison using the recommended amounts of siRNA and reagent for each reagent?

this part is confusing to me.

Thanks!

Just a question I haven't really fixed yet, how do you plan to check if your siRNA is inside the cells or just stuck to the membrane? Confocal microscopy? Has anyone done that before?

if you used synthetic siRNA for transfection, don't forget to rule out IFN effect which is also key to the inhibitory effect. We have done similar experiments to compare different transfection reagents on synthetic siRNA transfection. What we found is the brand name doesn't necessarily guarantee your higher silencing. One reagent, Gencarrier-1, stands out among others in transfection efficiency, knockdown effect, IFN effect and price.

Another thing is use 24, 12, or 6-well. The efficiency on 96-well is low.

Hi,

How would you identify which type of interferon you're dealing with? I doing a similar experiment.

Thanks

LNL



if you used synthetic siRNA for transfection, don't forget to rule out IFN effect which is also key to the inhibitory effect. We have done similar experiments to compare different transfection reagents on synthetic siRNA transfection. What we found is the brand name doesn't necessarily guarantee your higher silencing. One reagent, Gencarrier-1, stands out among others in transfection efficiency, knockdown effect, IFN effect and price.

Another thing is use 24, 12, or 6-well. The efficiency on 96-well is low.

IFN-gamma.
It acts through PKR (double-strand RNA-dependent kinase), eIF2 to shut off general cellular translation machinery (as a way to inhibit virus infection which take over host transcrition/translation machinery during infection).