I have a question about transfection method with siRNA and transfection reagent.
How can we explain the best transfection dose with 60 pmol among different doses (20, 40, 60, 80)? Why did not 80 pmol transfect better?
AND also How can we explain the best transfection time that is 48 h among other times (24, 48, 72 h)?
Personally, I use the Stemfect reagent for siRNA transfection. In my experience, knockdown of target genes could be detected as soon as 24 hours post transfection but a maximal knockdown is achieved after 48-72 hours.
I guess that the decline in knockdown after longer incubation times is related to degradation of siRNA molecules transfected to the cell.
Regarding dosage we have to remember that siRNA molecules are integrated to RISC complexes, which may be the limiting factor in regard to dosage (But that's just a guess. other factors must also have a significant impact).
Dear Masoumeh,
You have not given a lot of information to give even a good guess for the problem. For example, what and how many cells were you transfecting? 80 pmol =~ 1.11µg of DNA, so the transfection alone could get toxic.
You may also get off-target toxicity and other issues
This paper might help:
Article Action and Reaction: The Biological Response to siRNA and It...
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