To validate a miRNA target mRNA can we do it in vitro by adding everything in a test tube? If not, what are the difficulties and how can we overcome this?
I kinda doubt you can do it in a test tube. The gold standard for miRNA target confirmation is 3' UTR analysis. I think some companies even sell 3' UTR luciferase plasmids to many mRNAs specifically for this purpose. Expression studies are commonly performed, but are not as specific since there can be off-target effects.
Use qPCR to detemrine the half-life of your miRNA. Induce and watch expression changes. Plenty of companies sell reagents for these manipulations.
IVT (in vitro translation system) is routine in some labs. If you add the necessary components belonging to the RNAi pathway implicated in the processing of your miRNA together with your template, then it is possible. But, I think it would be not worthy to perform this for the purpose of validation unless you want to study some dynamics of the process.
I kinda doubt you can do it in a test tube. The gold standard for miRNA target confirmation is 3' UTR analysis. I think some companies even sell 3' UTR luciferase plasmids to many mRNAs specifically for this purpose. Expression studies are commonly performed, but are not as specific since there can be off-target effects.
Use qPCR to detemrine the half-life of your miRNA. Induce and watch expression changes. Plenty of companies sell reagents for these manipulations.
Hi, I agree with Jennifer, the best way is to check in repoter assay if there is interaction miR/3' UTR, so many plasmids are avaible like pmiRGlo (with two luciferases in the same plasmid) or others with only one luciferase. After if you have a cell-line expressing your gene of interest, a transfection with your miR and westernblotting can help you to validate your results
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