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The questions are what delivery route are you going to use--local or systemic delivery. If it is local, it should not be a big deal. If it is systemic, you have to find a good delivery agent that can bring the miRNA to its target tissue or organ. Regardless of delivery route, you have to prove first that your miRNA reaches its target tissue and internalized by target cells, 2nd, it suppresses its target genes.
#146172 microRNA validation in vivo
Posted
on 02 December 2012 - 12:35 PM
#139673 validation of microRNA target gene
Posted
on 20 August 2012 - 03:26 AM
Another test (but requires some work and maybe is not necessary) is to build a miRNA spongue to your candidate miRNA and check what happens with your candidate gene when the spongue is transfected into cells.
#139333 validation of microRNA target gene
Posted
on 12 August 2012 - 01:09 PM
The downregulation of eppty vector may be resulted from off-target effect of the miRNA or a response to the transfection. Strictly speaking, without showing that your miRNA has no effect on a gene lacking its target cannot support your conclusion that the target you found is a bona fide target.
A few questions:
What is your empty vector? luciferase only without the target?
Did your control miRNA mimics also downregulate empty vector?
I believe the standard control for such assay is to mutate the target (especially the seed region) which has been cloned into the 3UTR of luciferase gene to see if the miRNA of interest can still downregulate the luciferase.
A few questions:
What is your empty vector? luciferase only without the target?
Did your control miRNA mimics also downregulate empty vector?
I believe the standard control for such assay is to mutate the target (especially the seed region) which has been cloned into the 3UTR of luciferase gene to see if the miRNA of interest can still downregulate the luciferase.
#139336 validation of microRNA target gene
Posted
on 12 August 2012 - 01:49 PM
I am sorry for the misunderstanding. If you already have the mutated target vector control, then you don't need to have a empty vector control. The mutation of seed sequence abolishes the inhibitory effect by the miRNA supports your conclusion that the target is real.
