I am trying to inhibit one miRNA in MDA-MB-231 breast cancer cells using mirVana inhibitors (Thermo Fisher) and lipofectamine. I see efficient transfection since I in parallel transfect a cel-mir-39 mimic as transfection control but when I detect the miRNA levels of my inhibited miRNA using Taman assays after two days, I actually detect an increase in miRNA levels! (My values in fold increase with respect to untreated cells are a)negative control inhibitor: 2x increase, b)using 15pmol target inhibitor: 15x increase, c)using 50pmol target inhibitor: also around 15x increase.
I am wondering:
Thank you so much for your help!
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!!!
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