I'm new to siRNA and so have a number of questions, but I'll start here:
1. Reading the postings on this part of the forum it would appear as though scientists are comparing different transfection reagents and using knockdown of either GAPDH etc or their target gene expression as a readout. Don't you optimize the transfection using fluorescently labelled siRNAs?
2. People quote transfection efficiencies of say 60% and then say that this gives them >80% knock down. This suggests that; (i) In transfected cells, 100% of the target gene's expression is inhibited, and (ii) expression of the target gene is also inhibited in cells that were not transfected. Is this the case?....or,
Is this because of a reduced viability of some of the non-transfected cells or because the transfection efficiency is being under estimated ( ie. whereas it might be possible for 10 molecules of duplex siRNA to inhibit some expression, it would not be possible to detect these if they were fluorescently labelled)?
Thanks for any answers to these questions.