May I know what is the best cell density after 24 hours for Lipofectamine RNAiMax reagent for microRNA transfection in shsy5y cell line? Is reverse transfection recomended?
Dear Peiling,
The amount of DNA and Lipofectamine depends on the cell line you are using. As you said, it is SH-SY5Y, try increasing the amount. Additionally, I would suggest that passage the cells before transfection for actively growing cells and seed the cells atleast at three different densities yielding to different confluencies - 60%, 75% and 90% to optimize best transfection.
Also try not to transfect the cells at less than 60% confluency.
I would not suggest reverse transfection if you are already using Lipofectamine.
I hope it helps.
Cheers!
Hi Ayushi Priyam,
Thanks for your comment. It is really useful for me. In addition, may I know is there any specific ratio as for lipofectamine RNA iMAX: miRNA quantity: cell number?
Dear Peiling,
Regarding the quantity of miRNA: cell number it is a case to be tested. I mean, you will need try do a dose curve response, if it is not have been published yet.
In my case, for example, I needed to transfect epithelial cells with a specific antimiR, which is have been published before my experiment, but in that article the authors used a concentration of 50 nM to do their experiments. But with another miRNA. When I did my experiment and tried 50 nM, it is not worked. So, I needed to test with 3 different concentrations (25, 50 and 75 nM), I found that the best concentration for my case was 75 nM. I don't know in what exponential velocity your cells increase, but in my case, after did the transfection, I needed wait for more 24 hours, before do the other experiments (i.e. miRNA extraction, total RNA extraction) could be done.
I hope that it helps you.
Hi Pedro Paulo Gattai,
Appreciated your suggestion. Just wondering, will you suggest reverse transfection or forward transfection from your experience?
All this while I am doing reverse transfection using miR-1 positive control, however, the best efficiency I has got so far is 30% from the western blot result.
For all my siRNA transfections I find around 70% works well. If confluency is too low I see much more toxicity due to the transfection... too high and the transfection efficiency can suffer!
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