Hi All, Do you add the lipofectamine miRNA mix to the media which the cells are already in or remove the media and just add the lipfectamine-mimic mix in OPti-MEM media. And I also need to know what range of incubation do you recommend. The manual recommends 1-3 days whereas some people transfect for a 4 hours and then replace with growth media for 24 hours after which they analyze the transfection level. Which protocol do you recommend?
Inas, as you are probably aware, there are many commercially available reagents that purport to successfully transfect cells with high efficiencies using different nucleic acid molecules. Perhaps you may want to search out a company that sells a regent(s) that was optimized for miRNA. Some may even have fluorescently tagged miRNA molecules that you can use to test the efficiency of transfection. Nevertheless, I find that transfection of small nucleic acid molecules is quite an efficient process no matter the transfection reagent used. That being said, I have not transfected with miRNA, but I have successfully transfected with siRNA using Lipofectamine LTX.
My protocol is as follows (for 1 well of a 12 well plate):
1. Add 0.5uL of 20uM siRNA Into 200 uL of RT OptiMEM - this will result in a 10nM siRNA concentration when added to the well. Mix well by vigorous flicking of tube.
2. Add 0.46uL of Plus Reagent. Mix well by vigorous flicking of tube. Incubate for 5 min at RT.
3. Add 2.3uL of LTX Reagent. Mix well by vigorous flicking of tube. Incubate for 30 min (but I have let it go up to 1hr - manufacturer says one can go up to 6hrs if needed) at RT.
4. Aspirate medium in well and wash cells with 1mL of antibiotic-free medium (this is the same medium you use to grow your cells, but without antibiotic).
5. (Important) Aspirate wash medium and add 800uL of antibiotic-free medium. This volume will result in a 10nM siRNA concentration when the DNA/Lipo mix is added ot the well.
6. After 30min, mix DNA/Lipo mix by vigorous flicking, then add 200uL of DNA/Lipo mix to cells dropwise. Gently mix by tilting and swirling the plate.
7. Place back into incubator. No need to change medium after a few hours. I have left the same mixture up to 96hrs on primary corneal endothelial cells without any obvious negative impact. The important thing here is to have the proper control.
I typically achieve greater than 80% knockdown of our targeted protein after 48hrs. I can't guarantee that this protocol will be optimal for you, so if I were you I would spend the time necessary to optimize your conditions.
Best.
Hi Ricardo, thank you so much for your valuable answer. I am sorry that I did not mention the reagents I will be using, it is lipofectamine RNAiMAX Reagent and mirVANA mimic miRNA from life tech. Thanks again :)
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