validated siRNA doesn't work - (Jun/18/2007 )
hi
this is one of those problems that doesn't seem to have a solution-- a cheap one, that is. i am trying to repress expression of a gene for which there exists a published (validated) siRNA sequence. this sequence shows approx. 90% silencing for 24-72 hours, with about 40-50% silencing after 96 hours, at least according to the papers. Several groups have used the same sequence, with success.
we ordered siRNA of the same sequence from our core facility, which didn't work. i tried various transfection reagents, protocols, cell lines (even the cell lines used in the literature) to no success. we then ordered siRNA from dharmacon, and as it turned out, the antisense strand ended up having two extra G bases at the 5' end. the sense strand was the published sequence. this siRNA does not work either, and i've tried it with several transfection reagents, protocols, etc. as a last resort, i'm going to try to use Ambion's pSILENCER shRNA vector.
any ideas as to why this siRNA might not be working? i've tried transfecting it with a positive control in the same experiment (siRNA targeted to another gene) and the positive control worked beautifully. 
thanks!
PS: has anyone used Clontech's Single-Vector Tet-regulated Knockdown system? Does it work well for you?
Does the protein in your experiments have the same
Is the siRNA sequence that is published for the same species that you are trying it our on.
Is the siRNA sequence that is published for the same species that you are trying it our on.
yep, human.
That's really weird. Usually any siRNA against a gene may work, let alone validated siRNAs. Are you measuring mRNA or protein expression? Sometimes, you may not see the same changes in protein as in mRNA. Why dos Dharmacon put two extra Gs on the 5' end of the guide strand? If you want to add something, it occurs to the 3' end of the guide strand and the passenger stand.
Unless the original paper used a shRNA construct to express the siRNA, I think it'll be more effort that what it's worth? Maybe it would be easier just to design a new siRNA yourself?
Maybe you have RNase contamination somewhere? In that case the shRNA could work!
Good luck!
Thanks for the responses, folks. i'm getting quite desperate at this point! we'll see what happens.
hi It seems that our problems are quite similar. Have you found the solution? If yes plz tell me...
I am quite disappointed and upset with my results and supervisor's pressure is also there. plz help....
I have bought siRNA starter kit and validated Plk1 siRNA from ambion. I transfected PC3 cells with GAPDH siRNA available in the kit using siPORT Neo FX and could observe knockdown upto 40% at protein level when measured GAPDH enzyme activity by KDalert GAPDH assay. When I transfected 25,000 PC3 cells with validated Plk1 siRNA at 100µM, 3µl siPORT Neo FX in 24 well plate, I couldnot observe any knockdown at RNA level (RT- PCR) after 48 h whereas validation results show that the same conditions have given 96% knockdown in Hela cells. I am very much confused why am I not getting results from prevalidated siRNA. Please tell me what should i do?
saish