I am not clear what is rule behind mirna and mRNA targeting pair,because each mirna target officially 100s of target.
In such cases how one will validate legitimate mirna mRNA target.
Assays concerning luciferase cloned utr are biased because in physiology no one knows which are actual pair of mirna mRNA influencing each other.
You could validate it by qPCR, miRNAs are quantifiable that way. You can show physical interactions by performing pull-down assays to show that your miRNA interacts with a specific mRNA. RNA-crosslinking assays, etc.
Good luck!
You can use cell lines and quantify the mRNA and then in a second experiment knock down the miRNA in the cell line and again measure the mRNA levels. Comparing both might give you an idea of potential relation. Basically you have to eliminate all other variables except the ones you are testing.
it depends on several factors:
1. how many matches and mismatches are present in the target sequences which can bind to the microRNA
2. Physical availability of the target sequences (binding of the proteins to mRNA or its secondary structures and expression level of mir and mRNA, etc.)
3.half life of the mRNA
...
the best way to check the function of a microRNA is knock it down and then check the expression of your gene of interest at RNA and protein level. To investigate the potential interacting targets, you can use pull down assay. I would not recommend use of UTR luciferase assay and such, due to artificial increase in the level of mir and also its target (the level of mir and UTRs in luciferase setup are normally much higher than physiological states which in return might increase the possibility of false positive results).
good luck!
Via using additional mechanisms, such as described in
Trends Cardiovasc Med. 2014 Apr;24(3):128-32. doi: 10.1016/j.tcm.2013.09.005.
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