I am new at this... would like to know if anyone can help me... Thanks in advance!
I am trying to do a transfection with Dharmacon accell siRNA protocol in both cord blood neutrophils and cell line HL60.
I am using 1e6cell/ml at 1uM/ul siRNA with accell delivery media without FBS for 48hrs. When using siCTRL, the transfection rate seems to be at 100% by flow cytometry. But I was unable to detect a reduction in mRNA production for my gene when checking with qPCR. Is my cell concentration too high? Do I need any additional transfection agent?
Are you using a smart pool or a single siRNA. Mix of four works better.
I'm using the smart pool, but doesnt seem to see any reduction in mRNA expression.
I had to wait for 72h to see an effect. The problem was that by then my cells (moDC) started to dye. You have to be careful about the culture conditions. My cells didn't like the lack of FBS and growth factors. I had do add growth factors after 24h and did a medium change after 48h.
I am facing the same problem, I am using neutrophil and they started to die by 24 hours. :(
You can wait and can report this to Dharmacon, they should help you further. Sometimes the pool fails, but individual siRNA show great knockdown.
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