A question on RNAi efficiency? - (Feb/27/2006 )

I encounted a problem about the efficiency of RNAi recently. My target gene expresses at a quite low level in regular cells. When I co-transfect the RNAi with a GFP-fused exogenous cDNA, RNAi could greatly reduced the GFP fused protein, at the same time about 100% knocked down the endogenous gene. However, without the addition of GFP-plasmid, RNAi only could only reduced the endogenous mRNA to about 50%.
Does anyone have such experience or be able to give an explaination. Thanks.

Yes, the GFP is fused to the target gene. Previously I have already checked the expression of the exogenous protein, by fluorescence and by western.
The result after RNA silence was also checked by QPCR and by western.

This is an interesting explainaiton. But if there is a negative feed back loop, the endogenous mRNA after cotransfection with both exogenous and control RNAi should had also been downregulated. But the result was not like that.

Hi future21th!

so, your problem is that your RNAi sequence is working much better on the GFP-fusion transcript than on your endogenous gene. Is this right so far?

I also had this experience several times with a lacZ-fusion transcript (the "ScreenIt" strategy from Invitrogen). Sometimes I got the same results with the fusion and the endogenous transcript, but sometimes it was completely different (as also in your case).
My interpretation of this discrepancy is that the secondary structure of the fusion-mRNA might be different to the endogenous one. This could make it easier / more difficult for the siRNA to access the mRNA resulting in different efficiencies.
Since it's your endogenous transcript you're interested in (I assume so), I would rather trust these results than the fusion stuff.

Now, I always test my RNAi sequences on the real transcript - either endogenous or expression vector (without any reporter fusion).

I hope this helps,
Christiane