I would like to transfect my cells with a plasmid for a fluorescent protein (classical membrane marker I normally transfect via electroporation) AND transfect a siRNA to silence another protein. What would be the best way to proceed, any advice? Thanks a lot!
You can transfect both of them at the same time if you want. You can also perform the transfection in series. The ideal method would be using a plasmid with both the GFP and an shRNA cassette. Then you would know that all of the green cells also had the shRNA.
Thank you very much for this answer! Indeed I was wondering if transfecting both would work, or if the series transfection was an option. Of course if I would have a fluorescent reporter in the siRNA it would be best, but I don't at the moment.
Regarding co-transfection, the thing is that there is a lipofectamine reagent specific for siRNA, and another specific for plasmid. But I imagine if I use the siRNA one, the double transfection will still work, I may have to adjust the ratios, and i can visualise whether the fluroescent protein expresses at least. Alternatively, I was wondering if to use electroporation simultaneously as I normally use this method for the fluorescent-protein plasmid.
I was also wondering whether I could do in series as you suggest, starting with siRNA transfection with lipofectamine, and further doing an electroporation next day or two days later. But I aws wondering whether my cells would be very affected by such treatment, they are MEFs. Thank you again!
I believe co-transfection shouldn't be an issue, but it will save you a lot of time and headache in the end if you use a single plasmid for both siRNA and fluorescent marker. You can get a single construct (ie. siRNA and GFP) for $85. https://www.abmgood.com/RNAi-shRNA-sirna-shrna-rnai-lentivirus.html
Thank you, I bought this smart pool mix of 4 siRNA, but I will also consider this option! Indeed such a construct would help a lot.
Hi Anabel. This is in general not going to be a problem. Co transfections are very common protocols, you can find protocols of it anywhere. The key is to have double lipofectamine agent for every 1ug of transfected total plasmid concentration.
Regards Pranav
I see, thanks a lot for the tip!! I will try co-transfection, and also serial transfection in these conditions!
Dear Pranav, one quick question, you meant have 2ul lipofectamine agent for 1 ug total DNA? Thanks!
I would also be greatful if someone had advices on the relative ug or pmol that should be co-transfected as siRNA recommendations are in pmol (can I just calculate back the ug and chose a ratio, for instance 1:1 or 1:2, or isn't it correct, as the MW of siRNA versus a 5-7kbp plasmids are very different in size??? Thank you!
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