validated siRNA not working plz help - (Mar/20/2008 )
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
Hello Saish,
It sounds like you'll likely end up screening some new siRNA sequences to find an effective one. Alternatively, you might try a Morpholino oligo. Untried Morpholino sequences produce a good knockdown about 75% of the time, so you might only need to try a single oligo. Morpholinos have longer recognition sequences and therefore much less modulation of off-target gene expression compared to siRNA.
Summerton J (2007). Morpholino, siRNA, and S-DNA Compared: Impact of Structure and Mechanism of Action on Off-Target Effects and Sequence Specificity. Med Chem. 7(7):651-660.
Available online: http://www.gene-tools.com/files/Summerton2...iRNAcompare.pdf
Good luck with your knockdown, I hope it gets easier from here.
It sounds like you'll likely end up screening some new siRNA sequences to find an effective one. Alternatively, you might try a Morpholino oligo. Untried Morpholino sequences produce a good knockdown about 75% of the time, so you might only need to try a single oligo. Morpholinos have longer recognition sequences and therefore much less modulation of off-target gene expression compared to siRNA.
Summerton J (2007). Morpholino, siRNA, and S-DNA Compared: Impact of Structure and Mechanism of Action on Off-Target Effects and Sequence Specificity. Med Chem. 7(7):651-660.
Available online: http://www.gene-tools.com/files/Summerton2...iRNAcompare.pdf
Good luck with your knockdown, I hope it gets easier from here.
Thanks a lot for such a quick reply. But before trying morpholino or some other siRNA, I really do need to know a few things because validated siRNAs are supposed to work.Before switching onto something else need to give an explanation to my supervisor . Has, the validated siRNA I had bought, got degraded? What are the reasons for siRNA degradation?I had tried reverse transfection method. Should I try preplating transfection? Are the two ways of transfection very different and can that be the reason for not getting results? plz help.
You don't you try calling the Ambion tech support? They have always been most helpful when I've contacted them.
Thanks for your response. actually i have mailed ambion but have not received any reply yet. Hopefully i recieve some favourable advice from them. am really upset right now. thanks anyways.