Hi Shirin,

probably over 90% of target sequences for miRNAs are in the 3'UTR of higher eukaryotic genes, the rest can be in exons or very rarely in 5'UTRs. Drosophila miRNAS are a bit more spread out in the gene sequence. If in your animal/plant model the genomic sequence or complete mRNA sequence is not yet known for the gene of interest, I would start with a 3' RACE, which is also much simpler.

In any case, just the sequence is very little evidence of miRNA function on an  mRNA. The seed sequence is only 7 nt long and you will probably find it many times in hundreds of genes, sometimes with more pairings downstream in the miRNA after a loop or a mismatching region, and this second annealing part is probably what regulates its actual binding to the mRNA, its sequestration in P bodies and its translational silencing or its degradation...

You will have to show the regulation with a reporter gene assay, which means fusing the 3'UTR sequence of your gene (or the exonic one, or the 5'UTR) to a luciferase CDS and verify that co-transfecting with the miRNA reduces the luciferase activity compared to no miRNA or a scramble miRNA. Will be even better to also mutate the 3'UTR from your gene in the miRNA binding site and show that the reporter gene activity is not reduced when co-transfecting with the miRNA...

Good luck.

Hello Shirin,

Normally you verify the effect of a miRNA on a mRNA by cloning the 3´UTR into the vector of a Luciferase Reporter Gene Assay, see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646406/

In addition, it is often good to perform a western blot to ensure a loss of protein abundance because if the miRNA has a non-perfect binding the mRNA stays intact and is just shifted into a hold status without degradation.

You can make a perfect verification when you apply antagomirs silencing the activity of your desired miRNA

http://www.ncbi.nlm.nih.gov/pubmed/22956143

http://www.ncbi.nlm.nih.gov/pubmed/16258535

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877768/

Different vendorss sell these oligos, here examples:

https://www.thermofisher.com/at/en/home/life-science/epigenetics-noncoding-rna-research/mirna-profiling-/ambion-anti-mir-inhibitors.html

http://www.creative-biogene.com/Services/MicroRNA-Agomir-Antagomir-Synthesis-Service.html

I hope this is a help for you and your project :-)

Regards

Hi Shirin,

probably over 90% of target sequences for miRNAs are in the 3'UTR of higher eukaryotic genes, the rest can be in exons or very rarely in 5'UTRs. Drosophila miRNAS are a bit more spread out in the gene sequence. If in your animal/plant model the genomic sequence or complete mRNA sequence is not yet known for the gene of interest, I would start with a 3' RACE, which is also much simpler.

In any case, just the sequence is very little evidence of miRNA function on an  mRNA. The seed sequence is only 7 nt long and you will probably find it many times in hundreds of genes, sometimes with more pairings downstream in the miRNA after a loop or a mismatching region, and this second annealing part is probably what regulates its actual binding to the mRNA, its sequestration in P bodies and its translational silencing or its degradation...

You will have to show the regulation with a reporter gene assay, which means fusing the 3'UTR sequence of your gene (or the exonic one, or the 5'UTR) to a luciferase CDS and verify that co-transfecting with the miRNA reduces the luciferase activity compared to no miRNA or a scramble miRNA. Will be even better to also mutate the 3'UTR from your gene in the miRNA binding site and show that the reporter gene activity is not reduced when co-transfecting with the miRNA...

Good luck.

Shirin,

       Because 5' RACE or 3' RACE are targeted to your specific gene and are also more complex reactions than straight quantitative PCR, it is difficult to use in a quantitative manner with an internal quantitative control, which I think is what you are after.  The approaches above are appropriate for confirming a direct role for a miRNA.  However, if you just want to first check that the mRNA levels change, you are much better off just doing a traditional quantitative PCR, preferably with an internal standard (GAPDH etc.) to measure the relative mRNA levels for your gene + or - the miRNA.

Thanks everyone for sharing your valuable opinions with me, I forgot to mention that I work in plants (Arabidopsis thaliana) and in plants, most of the miRNAs alter the expression of their targets by cleavage of the target mRNA. That's why I chose 5' RACE (actually I have seen a couple of papers using that mehtod).

some examples :

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231765/

http://science.sciencemag.org/content/297/5589/2053

And I figured from your replies, that for further confirmation I should use GFP/GUS fused vectors and real-time PCR as well.

Thank you so much again :)

Hi Shirin, I think you might be mixing things up a little bit... at least from what I see from the two references you mention. The first one only refers to pri-miRNA processing: since mutating processing enzymes alters the precision of the processing, the 5'RACE on miRNAs was needed to demonstrate this imprecise processing. The second paper refers to one possible situation in miRNA regulation, the case when the miRNA is fully complementary to the mRNA sequence, which is now since many years well known to produce mRNA cleavage and then degradation (but of course the Science paper is from 2002, still in the pioneering period of miRNA research...). You are right that in plants this mechanisms appears to be quite widespread, still there is also a lot of translational regulation going on, although less explored, so that little is know on how the two pathways are regulated, if there is any switch between the two, as well as how much complementarity is needed for the translational silencing to occur (I don't want to preach in your specific field, but maybe a more up to date review might not do harm...).

Anyway, if you know the binding site of your miRNA on your mRNA, you can of course design a 5' RACE or 5' primer extension to try and find if your mRNA is cleaved at all. You could as well, as suggested by Prescott, run a qPCR with primers spanning the binding site of the miRNA in samples where the miRNA is over-expressed or depleted, if this is feasible. But if you should not find anything, you still have the possibility to measure the protein product over-expressing or depleting the miRNA and highlight a translational control. And a fusion reporter assay, as you already realized, with the mutation of the binding site, would help you confirming if the miRNA is really effective or not...

Good luck.

http://www.plantcell.org/content/25/7/2383.long

Agree with Shirin. 5' RACE should be enough after quantification by qPCR. If you have abundant of targets, degradome sequencing might be another option.

Hello again :)

Thank you so much Dr. @Pietro Pilo Boyl and @Abdul Fatah, I enjoyed reading your replies. I went through all of the replies again and I also checked the links you have sent me. I actually agree with you all, That's certainly the way to run such an experiment. The thing is, I only ran into this idea a couple of days ago while I was analysing my sequencing results. So i thought why not? I'll do a quick check and see if this mRNA has any cleavage site at all (as the first clue to go on further). That's why I was looking for a quick and non-expensive way to check it first.

Have a good day everyone :)