help with choosing model system for optimization of transfection of siRNA - and best deal for positive control? (Jun/21/2006 )

I am working on transfection of siRNA and would like to hear from experts here on which model system to choose for testing siRNA transfection.

I understand that co-transfection of a reporter gene with siRNA sometimes gives false % of inhibition because siRNA and reporter gene usually enter the same target cell; it is harder to inhibit an endogenous target, because the heterogeneous nature of transfection within a cell population, and not all cells receive the effective dose of transfected siRNA.

So I want to test transfection efficiency on either stably transfected cell lines with luciferase or EGFP as target sequences, or an endogenous host mRNA as the target. It will be easier for me to measure the inhibitory effects using reporters than choosing an endogenous mRNA target, which I need to do RT-PCR or western blot to measure the effect. Any suggestions which way to go?

My questions are 1) is expression from CMV promoter too strong to be blocked effectively by siRNA in stable cell lines? 2) Can you tell me which company sells positive control siRNA that is the best deal? Thanks in advance for suggestions.

I have tested mCMV- murine CMV, and the expression is quite strong to be blocked significantly by siRNA even if u have a good siRNA sequence.

We silence our gene in stable cell lines (gene of interest under the control of CMV promoter) and it works.
For the company, I can tell you that we use Ambion because the siRNA are good. I know that SantaCruz is also making siRNA but I don't know how good they are.
Don't confuse transfection efficiency and silencing efficiency: they are two different things! If you only want to test transfection efficiency, I can suggest to use a labelled siRNA to see how many cells got it: it's easier than looking at the expression of the gene of interst. If you want to check the silencing efficiency, then you don't have any other way than looking at the expression.

We got 80% silencing ca.