Hello everyone,
I am an researcher who work with huvec.
Several days ago, I did siRNA transfection on huvec to see whether
my gene of interest really contributes the migration of huvec.
(I have hypothesized that knockdown of my gene of interest would
slow down the rate of huvec migration)
I got results as what I expected several times, but lately,
I got result that does not match with my hypothesis.
I concluded that insufficient knockdown is the cause of such result
because I confirmed only slight knockdown in mRNA level.
(From real-time pcr, only 50% reduction was detected from my data)
I want to get any other or related advices on my unexpected result.
Thank you!
Check your reagents Lee, if your siRNA construct worked earlier maybe it has degraded and that's the reason your assay is no longer working well
Check the eficiency of transfection. May be you have only 50% transfected cells. Check the expression of the target gene depends on the silencing, some cells upload their espression profiles upon siRNA transfection.
Two things you need to check, first is the transfection efficiency, you may either increase the siRNA amount or increase the transfection reagent if the cell line is difficult to transfect. Second is the transfection reagent. Please consider using less toxic transfection reagents, the poor results may due to the death of the transfected cells.
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