I must transfect miR-145 mature to MCF-7 cell line.
What is the most effective method for transfection of Micro-RNA?
What is the best protocol?
Hi Mohammed, you can buy mimics of this particular microRNA from Life Tech and use their Lipofectamine RNAiMAX reagent for transfection into your destination cells. Check their website for the detailed protocol.
I have obtained high efficiency transfections with siPORT NeoFX in the past (70-80% transfection efficiency with human airway smooth muscle cells which are historically hard to transfect with high efficiency). The secret is to perform the transfection in suspension (sometimes called reverse transfection). Trypsinize your cells to get them in suspension at your desired cell concentration then add your transfection mixture (miRNA mimic complexed with transfection reagent) to the cell suspension (Typically combine 1 part cell suspension and 1 part transfection mixture) in a conical tube. Gently mix the resulting mixture by inversion a few times (the trick is to mix adequately enough to properly mix everything but not too roughly to damage cells) then immediately plate on your culture vessel. Incubate the cells for up to 24 hours in the transfection mixture then change out media with fresh culture media then either wait additional time or proceed with treatments.
During transfection I recommend OPTIMEM for diluting siPORT, miRNA mimics, etc...
In my experience the cells are more viable if they are resuspended in media with serum but make sure they are resuspended such that the number of cells/mL is extremely high.
30 nM final concentration of miRidian mimics seems to yield miRNA levels a few hundred-fold higher than endogenous levels and usually works for studying function but I recommend setting up an initial dose response to determine the optimal concentration because reviewers for probably request this.
miRidian mimics can be measured by qPCR to verify successful transfection.
Tex615-fluorescently labeled negative control siRNA from IDT can be used as a positive control for transfection as well to determine transfection efficiency.
Let me know if you need additional help. I just published two papers using this protocol and it worked great.
siRNA-Mate+lipofectamine as a transfection reagent and miR-145 mimics
I tried with phospho transfection but on MCF7 is really toxic, then I switch to Lipofectamin and it was ok!
Use Fugene HD. This is the safest. You can transfect both in presence or absence of serum. Just follow the instruction in Promega website. It is the best so far to me.
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