Subsequent DNA and siRNA transfection - (Apr/09/2014 )
I need to first transfect MCF-7 cells with plasmid DNA and subsequently divide the transfected population in two and perform two separate siRNA transfections. Does anyone have exprerience with this? I am afraid my siRNA transfection efficiency will be low. Can you think of any additional pitfalls or solutions?
You could make a stable cell line with the DNA you are transfecting in, then work on the siRNAs separately.
We have done transfection with plasmid and siRNA. We transfect them either sequentially (first plasmid and then siRNA as you planned) or in combination. Transfection efficiency is not a big issue.
hello both, thank you for the answers.
Bob1, unfortunately this is not an option. I already have shRNA stables but this particular KD affects cell proliferation and the population that did grow out is very different from the parental cells in unwanted ways!!!!
pcrman, thank you for the encouraging words. What siRNA transfection reagents do you use?
So you are doing a shRNA KD via plasmid and then KD by siRNA as well?
Erm... no! I did the shRNA to simplify things... it didn't work... I went back to the siRNA :)
OK - in that case making a stable line and doing the siRNAs after this would work just fine. Making the line takes a fair bit of time though.
We mostly use invitrogen's RNAiMax which has low toxicity.
Seconded about the RNAiMax - it is excellent.
for plasmid transfection of MCF-7 cells my colleagues use Viromer RED and Viromer BLUE for siRNA. Viromers were developed from Lipocalyx (www.lipocalyx.de).
Viromers are synthetic polymers zero in charge and very gentle to cells. Due to their active endosome escape mechanism my colleagues get higher transfection efficiency compared to standard reagents.
Please find attached some exemplary data.