In an miRNA overexpression experiment, say miR-17, with mimic transfected at 25 nM, is it possible that the effect is achieved through targeting of mRNA that are actually targets of miRNA from the same family and not of miR-17 itself?
since the seed sequences of miRNAs of the same family are same ...it is possible that if miRNA is introduced at higher concentrations it can bind to low affinity targets and hence some of the target of the same family ... but i am not sure which system are you talking about ?...is it in vitro cells or animal model systems where you are usning 25nM for miRNA overexpression ?
yeah sorry, I didn't specify. It's a cell line of breast cancer, MDA-MB-231.
Are you able to look at the affected mRNAs by microarray or RNA-seq? This would allow you to determine whether you're getting off-target effects.
Hello Chiara,
I think that it could be possible that you miRNA mimic target more efficiently another mRNAs that are targets of this miRNA family. miRNA of the same family could bind with different efficiency the same targets, an example of this fact:
Marco A, Hooks K, Griffiths-Jones S. Evolution and function of the extended miR-2 microRNA family. RNA Biol 2012;9:242-8
You have to take into account that miRNA and mRNA are not expressed always at the same time and it is possible that your effect in cell culture couldn't be possible in vivo. I mean, your miRNA maybe it is not expressed at the same time than this other mRNA targets. It is possible that your miRNA targets more efficiently the other mRNA targets of this family of miRNA, but the expression of this miRNA and mRNA in vivo & at the same time doesn't occur.
Hello! I think the effects will be complicated if you use a two-strand miRNA mimic. On the one hand, the miRNA will regulate the targets of its family , although the efficiency may be different within different members. On the other hand, the second strand will served as one siRNA and introduce off-target effect, so i recommend you pre-miRNA to use. In addition, the amount of miRNA transfected to cells is important to be considered, for the expression level of miRNA in the in vitro experiment seems too higher thant its physiological level.
In a simplified (over-simplified?) world, the over-expression of a microRNA of a given family will affect the targets of the entire family, following the targetScan predictions, based on the seed. In effect, in a fraction of these targets, there will be specificity of some family members respect to others, due to extended complementary beyond the seed.
In high-throughput screening assays members of the same family tend to give similar results. So it stands to reason that they effect very similar targets. The seed (and targetscan) is no longer considered the sole determinant of targeting but it does have a big effect. I think that the mimics are designed so that only one strand should have an effect
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