I'm optimizing my siRNA transfection with Lipofectamine 2000. I used 40-160 nM of Dharmacon siRNA and the ratio of Lipofectamine:siRNA is 10 ul for 10 pmol. I let the cells grow for 24h after transfection and harvested them for western blot and qPCR. But both show no change between control and transfected, even within concentrations.
Does anybody have experience with this cell line for transfection? Should I let them grow for 48h instead? And any suggestion for the amount of Lipofectamine? Thanks
Based on the experimental details you provided, it is likely that the ratio between the Lipofectamine and siRNA may not have been optimum. However, if you did not see any change between control and siRNA transfected cells, I believe that siRNA may not be authenticated for your gene/protein of interest. Therefore, keeping transfected cells for 48 h is not likely to change the outcome. The best approach would be to optimize the ratio and authenticate the siRNA.
Thank you. Actually I saw change in mRNA and protein level by 50% reduction when I transfected by electroporation. I just want to switch to lipofectamine to reduce the cell death.
Hello Nguyen,
Have you tested you cells for Mycoplasma contamination, this is a very good reason why transfection did not work.
See in this chat what answers we have discussed there:
https://www.researchgate.net/post/Has_anyone_performed_a_knockdown_via_siRNA_in_primary_keratinocytes#56e1e93d615e279bdf36b281
Hi Volker,
Thanks for your suggestion. It's actually my concern too. Can you tell me which kit have u used for Mycoplasma detection since I've never used it before
I have used an Applichem Kit bit maybe the order number is different in the states.
https://www.applichem.com/en/shop/product-detail/as/pcr-mycoplasmen-testkit/
The kit worked very good and very fast to receive a result.
Hi,
We wait 48 hours for gene expression experiments and 72h for protein analysis, the KD could be tested at 24h as this is the first step, but if you want to see the effect of this KD on the gene expression of other genes or even in proteins, this should be done at least at 48h.
I think is not a matter of contamination or toxicity, that could be too with lipofectamine but less than with electroporation.
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