double siRNA knockdown? - procedure for simultaneous silencing (Apr/23/2008 )

This paper used injection instead of transfection,
besides, it's oligos but not double stranded siRNA,
the intracellular mechanism will be very different

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Yes, as I had already posted suggesting that single-stranded antisense might be a better strategy for a multiple knockdown I did not repeat that when posting the link. The mechanism of knockdown does differ, the Morpholino activity is independent of RISC or RNase-H. The transfection mechanism for these oligos differs as well; while the cited paper was for an embryonic microinjection, Morpholinos are usually transfected into cells using an endosomal release agent. The endosomal release agent does not complex with the oligos, allowing the concentration of oligo to be varied independently of the concentration of endosomal release agent. Since the oligos are delivered uncomplexed, delivery of oligo mixtures is feasible so long as the oligos are not cross-complementary.

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I tried to transfect 3 different siRNA together and eventually, the real-time PCR results show only one of the genes was knocked down. And the biologic effect is the same as a single knockdown of that gene too.

Sequential knockdown then?

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Thanks for keeping me posted. Its good to know that the results seem to point to the idea that sequential would be best. Curious though as to how the cell selected which of your three siRNA to knockdown? I am heavily leaning to the idea of sequential knockdown as well. Three seems ambitious and wouldn't you lose the transient (if it is transient) effect of the knockdown? Unless you transfected every 12 hours or so.

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Hi did u try the sequential knockdown..let me know hat happened...i am doing the combined experiment now , i am trying to knockdown 2 genes in lung cancer cell line. I have mixed them half and half and get to final concentration 100nm , lets see what happens.
thanks:)

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so we just did a sequential rnai of two genes with a transfection one day then another on the following day. We actually did have pretty good success doing it this way. As I have said previously we use the lipid based transfection procedure and it seems to work. Hopefully we can continue to get good results. keep u updated

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I think it is possible, as in my lab, my mentor did the double transfection of Ezh2 and E2F1 for lung cancer cells.

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Ironically we have been working with E2F1 RNAi as well. We actually have had pretty good success with the sequential knockdown. We knockdown our primary gene of interest then 24 hours later knockdown E2F1 then 24 hours after that we look at cell number and apoptosis by western. Have you been doing something similar sonken?

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