Dear All,
For quite a long time I am doing siRNA with different genes.
Lately I found out an interestin phenomena.
I am looking for the effect of kd of gene A on protein B. It seems that depending upon the amount of kd that I am getting the effect on protein B is changing. Reported to this effect is when you kd A B is increaing. I found that if the kd is ~40-50% I am getting an increase in B but if the kd is lower or higher 10-30% or 80-90% the effect is the opposite.
It is quite surprising.
Did any one got something similar?
Looking forward for a disscution in this topic.
Guy

Do you expect any direct regulatory interactions between A and B (e.g. A represses B)?

It is known that knockdown of a given sequence by siRNA will modulate the expression of up to hundreds of off-target genes (Scacheri et al, below).

It is likely that in some cases off-target gene modulation is due to direct off-target expression suppression by the siRNA, while in other cases gene expression modulation would be expected as regulatory consequences of the direct knockdowns; that is, you target gene A, which represses expression of gene B through off-target siRNA interaction with its mRNA, and as a consequence gene C might be upregulated in response to the suppression of B (in this case a regulatory effect rather than a direct siRNA effect). To restate this, even though A and C have no direct regulatory link, C can still vary as a consequence of regulatory control in response to off-target knockdown of B.

Here are some citations of papers addressing the off-target gene modulation of siRNA:

Comparison of siRNA-induced off-target RNA and protein effects. Aleman LM, Doench J, Sharp PA. RNA. 2007 Jan 19; [Epub ahead of print]

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A. Nat Methods. 2006 Mar;3(3):199-204.

Nonspecific, concentration-dependant stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). Persengiev SP, Zhu X and Green M. RNA 2004; 10:12-18.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.