I've also been having problems with using a mimic for that same microRNA, whereby I've not been seeing the expected changes in expression of known targets of this microRNA.
I'm mainly looking for any tips and tricks more experienced people might have for achieving better results with these techniques.
Thanks!
Is this a lentiviral transduction or just a transient transfection?
Need more information. What amount did you use per transfection? In vivo or in vitro? Which company? Please provide as much detail as possible.
First tip - to use the "positive" controls in parallel which were shown to have expected effects in YOUR cellular system. If that works - go further with your miRNA of interest..
I guess that lipofectamine RNAi MAX was optimized for small RNAs transfections. maybe it would be better to use this reagent for cells that are more difficult to transfect. However, I used lipofectamine 2000 for plasmid plus microRNA cotrasfections, obtaining good results.
how do you check the silencing of the target microRNA? I guess it's better to check that also, before seeing if there is any change of expression of its targets.
Finally, might it be possible that the effect of the microRNA would be visible at the protein level instead of mRNA expression level?
What type of cells are you using? Because cell type is also an important factor in transfection. Some of the cells hardly transfected with usual transfection kits. For example I'm using an electroporator for suspension cells. I always check my results with western blot. Planing time course experiments will help.
Hello Hayley
I would like know some question like:
What type of confidence is your known gene to test your experiment? It is predicted by in silico analysis or experimentally?
Your experiment is based in the same cell type that your reference or it is different.
Your cell culture is cell line or primary cell
Please provide as much detail as possible
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