I want to do a shRNA transfection experiment, but there are too many shRNAs to be tested.
So, except for virus particle vectors, i wanna add only shRNA vector using lipofectamine2000.
Transfected cells (HEK293cell) will be harvested 2-3 days later, and qRT-PCR or western blot will be to be performed to check knock down efficacy.
and
I'm not going to use the shRNA vector that is not knocked down in this experiment.
Is it possible? ( Could I reduce the number of shRNAs I test with this experiment?)
Hi Hansong Chae,
excuse me if I don´t understand your question. Do you have different shRNA sequences for one target gene? Do you want to test if your shRNA sequence is effective in knocking-down its target expression? And you want to do it without transduction?
The "shRNA vector that is not knocked down", is that a negative control? (vector without shRNA or vector with an shRNA without no target in HEK cells).
As you may already know, it is usually easy to transfect HEK293 cells. If you know that your gene(s)/protein(s) of interest is(are) expressed in these cells at a sufficient level to detect changes, I think it may be ok to check your shRNA efficiency by transfection. However, I also think it would be important to include some controls that let you know that everything is working well.
Since the transfection efficiency is very high for 293 cells, the transfection of shRNA plasmid is ok. I am not sure whether 2 or 3 days is sufficient to knock down the mRNA/protein level.
Matías Gabrielli Yes, I have several shRNA for one target gene, and I wanna perform pilot test to decrease the number of shRNA tests.
for pilot shRNA tests, does it okay only perform lipo+shRNA step to check shRNA working without transduction?
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