Transfected copies per cell - (Nov/12/2008 )
Here is a question from a seminar I presented earlier in the week:
"So if you have a high population of transfected cells in your culture system, then how many copies of your siRNA are going in to each cell?"
This one kind of threw me for a loop! I know in my system I see about 95% of my cells end up positive for an Alexa labeled siRNA molecule, but is there a way to determine the copy number that each cell takes up??? I am using synthetic dsRNA molecules transfecting with HiperFect by Qiagen. I typically see around 95% positively transfected cells and get around 50% knockdown of my protein.
Hmm, the only thing that I can think of (and I don't think you would want to do it anyway as I think it will be pretty difficult) is some kind of qRT-PCR to check copy number before and after transfection and see how many copies are "missing" (from the medium - I guess you would do an EtOH precipitation of the medium) and then use the number of cells transfected to get a rough idea of copies/cell. Otherwise I have no idea!