Question about the control for an RNAi Experiment - (Dec/21/2009 )

I'm thinking what control should be included in a regular RNAi experiment to make the data validate (to be included in thesis/publication)? Basically, there are three controls which can be used:

1. normal, untreated cell
2. cell treated with transfection reagent only
3. cell transfected with validated negative control siRNA (transfection reagent+ nc siRNA)

Different paper reported data with different control, some papers used 1 & 3, while some used 2 & 3.

can somebody tell me which control to be included, so that relative density (relative remaining protein expression) can be carried out? To which control should I compare with?

Thank a lot!

All three to be on the safe side

The transfection reagent could cause phenotypic changes that would be independent of the siRNA. In this case, you would see the phenotype in 2,3, and gene of interest sample but not in 1

The presence of the siRNA could cause an off-targeting effect. This would cause the phenotype in only 3 and gene of interest sample (but not 1 or 2).

Each control is for a different potential problem so each has a place in the experiment.

Thank you for your reply!

If so, I plan to do 1 & 3 only. I think it is sufficient enough to validate the effect of my particular siRNA, no?

It will be fine as long as you don't see off-targeting effects with your non-targeting control and sample. If you do see an off-targeting effect, you will need to do the transfection reagent control to figure out the source of the problem.