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how to confirm miRNA knockdown?

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yuut

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I realized no matter whether LNA, or O-methyl or morpholino is used to inhibit miRNA, no body use qRT-PCR to show the knockdown effect. Why everyone does Northern? What is the reason there? I though qRT-PCR (like Taqman microRNA assay) is a lot more sensitive and easier than Northern?

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 Posted Jan 11, 2008, 2:40 AM
jonmoulton

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In the case of Morpholinos, the oligos do not degrade their target RNA so qRT-PCR isn't a good assay for their activity. If you look for the miRNA by qRT-PCR you will likely find it, but it did not have activity if a Morpholino was bound before you heated the heteroduplex and denatured it in the PCR cycle. If the Morpholino is targeted to successfully inhibit miRNA maturation, you would see the heavier precusor RNA accumulate by Northern (you wouldn't detect this by qRT-PCR, since you won't get target mass information from this assay).

For more discussion of using Northerns to detect buildup of immature miRNA, see:
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development. PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]
http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371%2Fjournal.pbio.0050203

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Jon D. Moulton, Ph.D. Gene Tools, LLC www.gene-tools.com

Posted Jan 17, 2008, 1:20 AM
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