When you are overexpressing miRNA, you are using endogenous processing machinery (drosa, dicer) to process pri- or pre-form of miRNA into mature form. When you are using miRNA mimic/artificial miRNA, you are giving it in mature form directly. This mimic does not require Drosa and Dicer precessing. In my experience, overexpressing miRNA through cloning pri-form did not work in all cell types. I guess endogenous processing might be hampered in this case. Overexpressing miRNA using CMV based vector leads to 100-200 fold induction of mature miRNA in my case which is sufficient to see the effect of a particular miRNA on its target gene. However, miRNA mimic can lead to several 1000 fold induction which might skew your conclusion. Because, it can promiscuously bind to any mRNA 3`-UTR that contains its binding site and downregulate that gene. But in real world, this miRNA may not regulate that particular gene in that particular cell. On the other hand, high concentration of mimic can also interfere with the function of other miRNAs that naturally target that genes through competition with binding sites in UTR or endogenous processing machinery. Alternate way is to suppress a particular miRNA through anti-miR and see the derepression of target mRNA. Now its your choice.

I am no expert about amiRNAs, but the overexpression of one native miRNA will likely cause overexpression of other genes/miRNas/lncRNAs... with the addition of one artificial miRNA you are sure that the only thing u are modulating is you miRNA of interest.

Good question, I think overexpression of specific miRNA probably will target one or more mRNA which will lead to down regulation of one or more proteins but the amiRNAs are designed to specifically target one mRNA. So, you can say, amiRNAs are more specific. 

When you are overexpressing miRNA, you are using endogenous processing machinery (drosa, dicer) to process pri- or pre-form of miRNA into mature form. When you are using miRNA mimic/artificial miRNA, you are giving it in mature form directly. This mimic does not require Drosa and Dicer precessing. In my experience, overexpressing miRNA through cloning pri-form did not work in all cell types. I guess endogenous processing might be hampered in this case. Overexpressing miRNA using CMV based vector leads to 100-200 fold induction of mature miRNA in my case which is sufficient to see the effect of a particular miRNA on its target gene. However, miRNA mimic can lead to several 1000 fold induction which might skew your conclusion. Because, it can promiscuously bind to any mRNA 3`-UTR that contains its binding site and downregulate that gene. But in real world, this miRNA may not regulate that particular gene in that particular cell. On the other hand, high concentration of mimic can also interfere with the function of other miRNAs that naturally target that genes through competition with binding sites in UTR or endogenous processing machinery. Alternate way is to suppress a particular miRNA through anti-miR and see the derepression of target mRNA. Now its your choice.

Hi Ruhul

What I mean by artificial miRNA is one with different miRNA precursor. If you see this paper, A new strategy for construction of artificial miRNA vectors in Arabidopsis, Planta, the authors have constructed an amiRNA vector with miR319 backbone. While comparing the results, they have WT, 35S:MIR319a and 35S:AMIR319a, with latter two having same phenotype. Also, the first paper by Schwab et al (Highly specific gene silencing by artificial miRNAs in Arabidopsis), plants with amiRNA construct were compared with loss of mutants for target genes.

Now, I understand that miRNA mimic has just the mature sequence and are not subject to DCL processing. But for overexpression under 35S and amiRNA, we are technically doing the same thing, ie., silencing of genes. So why design new amiRNA vectors for something that we can be do by simple overexpression!

Please correct me if I am wrong somewhere.

I thought you are talking about mimic and mammalian cell. The scenario in mammalian and plant is slightly different. mammalian miRNA can suppress multiple genes because mRNA targeting does not require perfect complementarity, partial matching in the seed region can suppress a particular mRNA. On the other hand, plant miRNAs has fewer targets which might be because of the requirement of perfect complementarity. In that case, natural miRNA can not suppress a group of mRNAs or homologs. In amiRNAs, you can target homologous genes by incorporating mismatches. Sometimes, miRNA-miRNA* (22nt) is entirely replaced with different miRNA/siRNA keeping the miRNA backbone intact. That way you can achieve tissue specific expression of your particular miRNA/siRNA. You can also incorporate multiple miRNAs as a polycistron in the same vector. When your focus is in single mRNA targeting, designing perfect complementary amiR might be helpful, if you want to target homologs or other related mRNAs, design in a different way. But just to see the effect of a particular natural miRNA, simple overexpression is enough I guess.

Over-expressing a particular miRNA and applying amiRNA mimic show the same effect on a specific gene. The methods you used affect the efficiency of it. 

Hi! Overexpression of miRNA  results in miRNA mimics, you can use it for example to restore phenotypes caused by downregulation of a native miRNA. The result will be that all the target trascripts of a specific miRNA will be regulated. AmiRNA on the other hand, gives the possibility to design miRNA against any gene/sequence of choice, you are not limited to the natural miRNA sequences. You can design  amiRNAs which do not exist in nature and use this approach to regulate genes or gene family for which no miRNA are available.

Both the approaches wouldn't necessarily result into the same extent of regulation.

Overexpressing a miRNA is a very tedious and time taking task but no less a fruitful one. for this you just insert the native precursor sequence of the desired miRNA into a vector and transform the plant once or for several generations to obtain stable miR-overexpressor line. you let the plant's own endogenous machinary to process the enitre thing and form a mature miRNA. it may or may not be up to your expectations due to very less or no mature miRNA produced.

however when an artificial miRNA technology is used, the precursor you are using is already known and tested to be high yielding i.e. it surely would give a mature miRNA sequence. thus the result will be according to your prediction. 

Endogenous miRNA are more or less complementary to the target genes but artificially designed miRNA are perfect complementary to the target gene and may cause prefect silencing or cleavage of target mRNA

Because all genes are not known to be regulated by miRNAs. Genes which are known targets of particular miRNAs can easily be down-regulated or up-regulated by overepxressing or suppressing their regulating miRNAs, respectively. While genes which are not known to be regulated by miRNAs, can be knocked down using RNAi. Now RNAi through hairpin-dsRNA have off-target effects. Therefore, people came up with amiRNA approach, which uses backbone of endogenous miRNA, where miRNA and miRNA* are replaced by amiRNA and amiRNA* to downregulate your gene of interest. amiRNAs use the same machinery as used by pre-miRNAs to produce mature miRNAs.