Hi Fernando,

Actually there may be several possible reasons: a) lipofectamine based upregulation (often commercial products contain info in compatibility on lipofectamine and cell lines); overload of miRNA during transfection due to TLR-mediated reaction.

In our experients we always used miRNA-free transfection as one of the negative controls.

Dear Fernando Lillo

The situation which you are going through, apart from observation and experimental setup, there are few thing you can reconsider and verify it once more

1. Transfection reagent: As you mentioned, you are using lipofectamine. In my study, I am successfully failed to transfect mimic/inhibitor & NC to same MDA-MB-231. Then I changed the transfection reagent to Polyplus Interferin, which is wonderful product and helped me to achieve successful transfection and results. Since like you, many reported the use of lipofectamine. here, in your case, I wish Lipofectamine is not a culprit.

2. Taqman for detection, even though it is good probe but my recommendation is recheck the Taqman compatibility and efficiency of miRNA detection in your context of whole miRNA detection process. (Other comments also suggested the same)

3. Your experimental setup is looking fine. The endogenous level of targeted miRNA in Untreated control say just cells and level (fold increase) of targeted miRNA after mimic transfection should be very clear.

4. It may sound odd , like mine other points above: BUT DOING THIS NOTHING HARM, WHY NOT YOU CHECK ONE-TWO POSITIVE TARGETS (mRNA level) OF YOUR TARGETED miRNA, and correlated with your upregulation-upon-inhibition results.

cheers!!!