Hello everyone,
I am trying to validate a microRNA target, I am simply new to this and I would like to know from those who already have experience, what reagents do I need and how could I validate it? I have read that the best way to validate it is through the luciferin-lucifersa method, can someone help me by providing me with the protocol or some references?
Thank you
Hi Rocio,
First, you should clone the sequence containing the predicted binding site(s) into a suitable vector (pmiRglo, for example). Usually, but not always, the sequence containing the binding sites is the 3'UTR of your target.
Then, you will cotransfect your cells with the reporter construct (and in parallel the empty reporter vector and the mimic of your microRNA or a scrambled version of your mimic (in alternative, you can somehow modulate your microRNA).
At the chosen time points you will process your samples to measure the luciferase activity in your samples. You will have to "normalize" your data using a second luciferase, such as Renilla, whose activity is not affected by the treatment, as its expression is under the control of a constitutive promoter.
Repeat the experiment to validate your observations.
If you find that modulating your microRNA is indeed changing luciferase activity, you can mutagenize the putative binding sites on your reporter construct and repeat the experiments as above, to show that if the recognition is impaired, modulation will not occur anymore.
Good luck!
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