I am working with a protein whose 3' UTR sequence is targeted by some of the miRNAs. I will perform luciferase reporter assay to be sure about that these miRNAs cut my mRNA's 3'UTR sequence.
But I am not sure about the control groups.
What I am trying to ask is that; Do I need to use 3'UTR mutants as well as miRNA mutants.
İn my design,
I use wt Over expressed miRNA + Target 3' UTR cloned plasmid
And as a negative control I use wt over expressed miRNA+ mock lusiferase vector.
I am not sure about using miRNA mutant. The fact that I will measure the lusiferase activity under miRNA activity that is compared to negative control I dont really think that I need miRNA mutant. Because the main idea is to validate that predicted miRNA cuts the mRNA or not.
But as I mentioned do I need to use a miRNA mutant or not. If yes ; would you mind to explain please?
Hi !
There are different necessary controls for this kind of test.
The first is indeed at the miR level. You have to test your wt miR and a negative miR which doesn't target the mRNA to verify that is not the (massive) overexpression of a miR in your system which disrupt something. The best is the miR mutant but you can also use a scramble version of your miR or use another well known miR which doesn't target your mRNA (but it is more risky as it targets another mRNAs). I also always test the empty expression vector to verify the impact of the transfection.
Secondly you have to test a mutant construct of your target. Indeed the overexpression of your miR could lead to a repression of your target but it doesn't tell you that is due to a direct activity of the miR. So I test my wt miR, mutant miR and empty vector on wt and mutant targets.
To finish, but it is the first step of a good luciferase reporter assay, you have to use a dual system with two luciferase vectors: the Firefly one with the modified 3'UTR and the unmodified renilla one which is the necessary internal control (transfection efficiency, washing ...) to normalize the Firefly activity.
Have a nice experiment and satisfying results!
Thomas
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If you have any other vectors like GAPDH or Beta Actin then you can also check that to make sure that your miRNA is targeting specifically only to your mRNA target.
FR
@ Farhan I dont really understant what do you really mean with GAPDH vector. Do you mean to clone GAPDH or Beta Actin's 3'UTR in to the lusiferase vector? I dont really agree with this idea actually the fact that in lusiferase assay it is not required to use an internal control gene.
@Thomas thanks for your answer, it guided me.
I decided to use this design;
Cel-miR-67 for negative miRNA control. This C.elegans miRNA has no mammalian homologus moreover has no target in mammalians
Special miRNA over expression contracts: that express my miRNA interest.
Gene of interest's 3'UTR: for normal experiment.
Gene of interest's 2 nucleotide mutant 3'UTR from miRNA binding site : For normalisation.
I have glomiRNAtarget vector with dual lusiferase. So I will have an other control to normalize lusiferase activity.
Thanks for your help. I am very pleasured for this answer.
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