Hi all, our lab is studying the role of a microRNA in cardioprotection. We want to observe what will happen if we remove this microRNA. We've tried to tranfect cells with antagomir/lipofectamin 2000 but the transfection efficiency was poor. The approach of using siRNA expression plasmid seems have potential. I am appreciated if you could suggest some protocol to do that? Thank you very much
Hi Nghiem, the transfection efficiency us often higher for smaller sequences such as inhibitors vs an expression plasmid thus if you are having issues with your transfection efficiency an expression plasmid may yield even lower efficiency. The one issue with the inhibitors is that you usually have to transfect them at high concentrations as they get degraded in the cell. In any case I would recommend that you first optimize your transfection efficiency and the conditions (unless you are using a lentiviral expression plasmid). This could be done for example with a luciferase reporter containing repeats of your MiR target sequence and performing a dose response curve with the target mimic/inhibitor. I prefer to use Fugene HD for single transfections and DharmaFect Duo for dual transfections. Feel free to email me if you have any further questions at [email protected]
What cell type are you using?
If your cells are adherent try using RNAiMAX instead of Lipo2000 it is more efficient and less cytotoxic.
then for transfecting plasmid we tried jetPEI on primary cells (endothelial and smooth muscle cells). The smaller the plasmid, the better the transfection rate. Lipo2000 and lipo3000 didn't work in our hands.
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