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How to design the 3UTR seq for luciferease Assay


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#1 SAN1017

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Posted 04 December 2009 - 09:06 PM

Dear All,

I'm working about the miRNA target 3UTR study. I found that some papers will clone the whole target gene 3UTR to the luciferase expression vector. However, some other papers will only clone a part of seq that contain the target binding site for the luciferase study. I would like to know which one is correct?
Besides, if i chose partial 3UTR region for the binding of miRNA, what is the criteria (like, how long of the seq) for the 3UTR seq so that the editor will accept the result?
Thanks all of miRNA professionals

San

#2 pcrman

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Posted 05 December 2009 - 10:15 AM

There are pros and cons cloning full-size 3'UTR or just the targets. Theoretically full-size 3'UTR can most reflect a natural situation because the flanking sequences and additional miR targets are kept intact. But the most commonly used strategy is cloning several copies of the predicted targets in tandem. This can be done by oligo cloning. It has the advantage of isolating the target from confounding factors, easy mutation of the seed sequences, easy demonstration of miR targeting effect.

If you choose partial 3UTR, I am not sure how long is enough. Given most people have used multiple copies of oligo targets, you can argue for any length.




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