I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium.
Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in growth medium? (Because serum works as a transfection inhibitors for some reagents...)
Hello,
Yes it's the right way.
The more important thing when you make your transfection master mix is, in the first time, to separately dilute RNA and lipofectamine with optiMEM ( Solution A = RNA + optiMEM and solution B = lipofectamine + optiMEM). Secondly, add A in B (drop by drop, no vortex) and incubate at room temperature (30 min with a plasmid, I'm not sure about RNA but I think at least for 5 or 10 min) before adding in growth medium.
Serum inhibit only liposome assembly, this is why it's so important to prepare transfection master mix in optiMEM and incubate sufficient time. After that, you can add your mix into medium supplemented with serum. However, for my part, I prefer use warm optiMEM. I incubate cells with optiMEM + transfection master mix during 6h (37*C, 5% CO2) and then replace the transfection medium by growth medium (without antibiotics).
Good luck.
Dear Clément Fage
Thank you for fast reply. Actually, I performed two times siRNA transfection and for both times I did the same. For the first time it worked perfectly and I had a clear reduction, but for the second time it didn't work!
I prepared solution A and B as you mentioned, then added this mix to the cells with growth medium+FCS.
For the first transfection, growth medium+FCS was so old (prepared 2months ago) but this medium was finished and for the second transfection I prepared the fresh medium+FCS. Because the second time it didn't work, I thought maybe fresh FCS worked as a inhibitor.
If I understood correctly, you prepare solution A and B and add to cells which they are in the optiMEM medium and after 6hr you replace this transfection medium with growth medium.
I plan to do it the same as you. I have a question, because I isolate protein after 48-72 hrs, if I replace this transfection reagent after 6hrs, new cells that appear after 1-2days can not uptake siRNA, how do you solve this problem? Maybe with second transfection in the next days?
_ Because the second time it didn't work, I thought maybe fresh FCS worked as a inhibitor.
It also might be due to a cells issue (confluence/contamination for example). You only failed once, so try again.
_ I have a question, because I isolate protein after 48-72 hrs, if I replace this transfection reagent after 6hrs, new cells that appear after 1-2days can not uptake siRNA, how do you solve this problem?
To be honest with you, I performed a lot of transfection but only with plasmids (influenza reverse genetics system, 8 plasmids in one cell to produce virus) never RNAi. In my protocol, I consider 6h as enough for the majority of liposomes to be adsorbed by cells. So, even if you let your transfection medium on cells during 2 or 3 days, I am not sure there still have a lot of liposomes. However you can increase the intake of RNA/lipofectamine or transfect a 80-85% confluence cells culture to decrease untransfected cells appearance.
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