From our lab experience, highly expressed miRNA in the cell are around 100-300 copies per cell. A 20nm transfection usually yields around 100000 copies of mimic miRNA per cell. You should be able to KO the luciferase vector as well as all the other targets of that miRNA without a problem. We usually see the largest KO around day two for some of difficult targets. You can add an antagomir control using a different concentrations to derepress your luciferase vector. Different concentrations should give different amounts of derepression of the luciferase

Indeed you might have some competition with the endogenous target. However there are several things you can do:

1. express your report with and without co-expressing with the miRNA.

2.I strongly suggest you to have as a negative control a altered UTR sequence that does not allow miRNA binding. If you see a decrease in LUC values when comparing your reporter with this negative control you can be more certain that the effect you are seeing is due to miRNA activity. Still in this system you need to be very accurate when reproducing the experiements and have a lot of replicates to be sure.

Hope it helps.

The luciferase assay is just a checkpoint in the microRNA targeting validation. As said you Cláudia, You need controls in order to be sure that luciferes expresion levels are diferent with and without the targeting microRNA and without the site in the UTR of the construct.

In the other hand, If you use LNAs you can not be sure that changing levels in your gene was due directly to the effect of the microRNA on the target, or by a posible motif involving both, target gene and microRNA.

you could do your experiment in a cell line that does not expresse your target mRNA, however each miRNA has many mRNA targets that no one yet knows, and that could compete with, if you also use miRNA mimics from 50 to 100 mM is so much that the competition of your mRNA should not be effective, usually I use miRNA mimics from 10 to 20 nM and they work good with luciferase reporter...

hope it helps

Thanks all very much for your responses. Targeted mutations in the UTR is indeed a stage I intend on performing in this experiment. The concentration of my mimics is usually 50 nM, but I might vary this with an optimisation control to see what's best. I'm aware of the different controls needed, but I still worry about a result that might be too subtle to detect.

@Rafal: I've indeed considered protecting the predicted target site with morpholinos. The disadvantage of this in my case is that my miRNA has been predicted to target two genes that co-regulate each other. This means I have to protect two sites, which brings along degrees of uncertainty. Do you by any chance have any experience with this yourself?

From our lab experience, highly expressed miRNA in the cell are around 100-300 copies per cell. A 20nm transfection usually yields around 100000 copies of mimic miRNA per cell. You should be able to KO the luciferase vector as well as all the other targets of that miRNA without a problem. We usually see the largest KO around day two for some of difficult targets. You can add an antagomir control using a different concentrations to derepress your luciferase vector. Different concentrations should give different amounts of derepression of the luciferase

It is a very good question Sebastian. I recently transfected some mimics and assayed the level of the mature miRNA using ABI taqman assays. The increase in the mature miRNA level was more than 50 fold for 50nM of transfected miRNA mimic. I believe that at such high concentrations there would be enough RISC complex to regulate both endogenous and exogenous targets.

@ Paul and Sanka. I've found similar results as you, the upregulation of the miRNA is indeed very high. Although the likelihood that competitive inhibition from endogenous mRNA is reduced by this, I was seeking to rule out this possibility.

Hello...

I was reading your question and answers and felt that maybe you could help me with my luciferase assay.

I'm doing luciferase assay in transfected cells with two differents alleles of a polymorphism. Both alleles are cotransfected with my microRNA from interest and a microRNA negative control. One allele has "seed site" from this microRNA and the other allele don't.

I found differente expression of my gene comparing the alleles + miRNA, but what I can not understand why my negative control miRNA is keeping different in the alleles comparison.

It should be the same gene expression in both alleles cotransfected with negative control, right? 

Anyone does have an idea what could be happening?

Thanks a lot!