double siRNA knockdown? - procedure for simultaneous silencing (Apr/23/2008 )
I was wondering how one would go about transfecting siRNA transiently into mammary epithelial carcinoma cells. Introducing single siRNA constructs is something we do all the time to silence one gene, however, is the protocol the same if we wish to silence two genes at the same time using two different siRNA complexes? Simply add both siRNA's to the cells at the same time? We use the lipid based protocol to introduce our siRNA into cells so any suggestions along these lines would be greatly appreciated, thanks.
The result is hard to predict. If you have one working siRNA for each gene and they work great when transfected separately. When combined, the two genes may not be equally knocked down, probably one gene won't be knocked down at all. I guess this has something to do with Ago2's preference for a certain configured dsRNA over another. The one favored by Ago2 can saturate Ago2 leaving the less favored one no chance of being processed by Ago2.
That is a very interesting point. I was wondering if you think doing one transfection then twenty four hours later doing another on the same cells? The genes we are targeting do no neccessarily act at the same time as one is downstream of the other so do you think this would produce more productive results.
You can just give it a try: either combinatory transfection or squential transfection. Only experiments can tell.
If you tie up the available Argonaute with the first siRNA, you might not find much Argonaute to receive the second. An alternative approach is to use a knockdown oligo that does not require Argonaute, such as single-stranded antisense.
I also want to do multiple siRNA transfection into the same cells to look for combined effect of knockdown
I have some doubt about sequential transfection as the efficiency depends on confluence of cells and it will definitely change in 24hrs, so the efficiency of the second transfection will be lowered or harder to control
Besides, I am not sure if back-to-back transfection will cause any mortality to the cells
I agree. I have not thought about the confluence issue but this is a good point. I do not however believe back to back transfection will directly cause cell mortalilty if using the appropriate reagent. But I will let you know how it goes!!
I am going to try to transfect 2 siRNA together in a single reaction and see what happens
Let me know how it goes. I still have not tried it yet but plan on in the near future.
Here's a published example of a quadruple knockdown.
http://www.pubmedcentral.nih.gov/articlere...bmedid=16360041