Dear colleagues,
I was wondering if someone have ever done such an experiment. I have successfully form an intact and compact cell monolayer with HUVEC cells and now I want to apply the protocol of transfection that I use for other cells to HUVEC. However, to form a barrier, I need high cells numbers and full confluency, whereas the transfection protocol suggest to use cells at 30-50%max confluency. Is it enough for this purpose to increase the amount of RNA and transfection agent (oligofectamine in my case)? In my case I should increase the amount 10 times and it seems to be too much.
What do you usually do?
Thanks!
It's very easy =)
Though the transfection works better when the cells are still growing=not confluent. We use Dharmafect 4 in HUVEC. I know from experience that you can transfect cells at higher density, but the efficiency will not be that high.Test the levels of you silenced gene after transfection at the density that you want and decide if the KD of your gene is enough for your purposes...
Also, many times, increasing the concentration of siRNA just causes off-target silencing, so be careful. I use a final concentration of siRNA of 25nM and, though with calcium phosphate I have tried up to 50nM, I would not go for 10x more...
Thank you Jennifer, it is exactly what I thought.
However in this way I will never be able to form a monolayer before the transfection effect disappears...how do you manage to do that?
In my case I am interested in producing exosomes when I knock down one protein. I checked the levels of gene expression of my protein at 24, 48, 72 and 96h post transfection and I saw that the RNA levels are knock down at all time points though the expression is slowly been restored through time. Also, I checked the protein levels at the same time points and though at 96h the RNA levels were almost the same, the protein levels were much more affected and the peptide was still very KD after 4 days...
Every protein is a world. Maybe you can try and transfect your cells just after they achieve confluence and you are lucky and your gene is effectively silenced... If there's is no bibliography available, try, and see if those results are good enough for you. Or you can prefer to use another approach. Not long ago I attended to a presentation by Exiqon where they presented a gapmers. These also KD proteins and that they (always keep in mind they were publicizing their own product) seem to be more stable over time....
I used Dermafect 4 and mu HUVEC were all dead few hours after transfection. I used few concentration one of them was 4 time less than their suggested concentration. And ideas? advice? help?
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