siRNA optimization - (May/09/2011 )
I designed two sets of siRNA for a tumor suppressor and I am planning to optimize it. I cotransfected 293s with my plamid (2.5ug) and various amounts of siRNA (100,500,1000pmols) using lipofectamine 2000. However, I have not got any lesser expression of my protein using these siRNAs.
Do you think, my siRNAs are not working? or maybe my experiment has some problems?
Thanks for help
What is the purpose of co-transfecting the plasmid?
The concentrations you used seem a bit low. You can try higher concentrations such as 10 - 50 nM.
How long did you transfect your cells? Have you analyzed mRNA expression of your gene? If you provide more detailed information, it will help.
So I transfected the cells with plasmid and siRNA in order to find out if my sirna can decrease the levels of overexpressed cells. I usually gather my samples after 24-48 hours. I have not analyzed the mRNA expression of my gene. I thought by co-transfection I can be sure that my cells are expressing my gene.
I think you can tweak several things:
Sequential transfection instead of cotransfection
Transfect longer than 48 hrs
Detect mRNA expression
Be ware that vector mediated overexpression could be hundreds or thusands folds, whether you could still see knockdown is questionable.
Thanks, Again I faced some other problems...
I can see a big band on Samples transfected with the plasmid only (positive control). however this band is decreased to untransfected levels(negative control) when I co transfected with my plasmid and scrambled siRNA! The same band is in sample cotransfected with plasmid and siRNA in the concentation of 100pmol/2ml. There is more intensity in samples cotransfected with plasmid and siRNA 500pmol/2ml and siRNA 1000pmol/2ml!!!!
How should I transfect sequentially? Should I transfect the cells with plasmid first and after 24 hours transfect them with siRNA? Is siRNA effects still detectable after 48 hours?
So my vector is around 5.4 Kb. Is it too big for siRNA to knock down?
Please help me...
You can first transfect plasmid, then the 2nd siRNA. RNAi action should last at least 5-7 days. RNAi has nothing to do with the size your mRNA or protein.