Hello! Could anybody suggest a transfection concentration for mir181-a? I have seen in papers people using around 50nM for transfection of mir181-a mimic, however I feel that such a high concentration may be toxic to the cells. Could you please share your experience with microRNA transfections?
This strongly depends on your type/source of mimic. However, 50 nM sounds ok (see different publications / using pubmed, google etc.).
Moreover, you could possibly try to test different conc. ranging from 5 - 100 nM, which should also work (e.g. 5, 10, 25, 50, 100 nM).
Hello! I fully agree with Andreas. 25 or 50nM for siRNA or miRNA are very usual concentrations. But testing a range from 5 to 100nM is always better.
If it can help you, please check the protocol for our Viromers BLUE/GREEN, on page 10 you have this kind of optimization guide, on page 13 a real dose-response curve. It's applicable to any other reagent.
https://viromer-transfection.com/wp-content/uploads/2015/10/transfection-protocol-Viromer-BLUE-06-15.pdf
I am currently using Lipofectamine RNAimax and Optimem as my Transfection Reagents. The manufacturer recommends 2x 250 uL of dilution medium. Does that include the amount of Lipofectamine and miRNA that I am adding or it just refers to the Optimum alone?
I assume you are working in 6-well. Then the 250µl mix you add on cells is made of 1/2 diluted miRNA and 1/2 diluted RNAiMax. The protocol recommend to prepare 300µl in total: 150µl of diluted siRNA (exactly it is 3µl siRNA into 147µl OptiMem) + 150µl of diluted RNAiMAX (exactly it is 9µl of RNAiMAX into 141µl of OptiMem). If you use 150µl instead of 147 and 141µl, it's peanut, it's the same...
I am currently using LNA MIRcury miRNA from QIAGEN. I have used different concentrations (from 1nm, 3nm, 5nm, 10nm, 30nm, 50nm). I am using 150uL of Opti-MEM + x uL of Mirna and 150 uL of Opti-MEM + 7.5 uL of lipofectamine. From there I do a 1:1 dilution as recommended by the manufacturer and from there I pipette 250 uL to the well. = Final volume equals to 2ml of media + 250uL of Transfection Reagent. I change the media 6h after transfection.
So far I could not have good efficiency (I am having around 30-40 transfection efficiency). Do you guys now what else could I do to optimize this?
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