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using mirbase?


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#1 Mr. Naive

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Posted 30 March 2010 - 09:18 AM

Hi folks, Someone please help me how to identify the function of a specific miRNA after identifying it, using microarray.

thanks.

#2 Jon Moulton

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Posted 30 March 2010 - 01:16 PM

There are many possible strategies. Here is one. Use a Morpholino oligo complementary to your pri-miRNA of interest (ideally a 25-mer spanning the guide sequence and extending slightly into the loop). Introduce the oligo into the cytosol of the cells, embryos or organism you wish to test. Wait a few days and harvest RNA from treated cells/embryos/organisms and untreated control cells/embryos/organisms, comparing the RNA from the treated and untreated cells/embryos/organism on microarrays.

For a specificity control, you might use a Morpholino targeting the guide strand and extending a bit into the loop. This may take longer to show an effect, as it will not bind to guide miRNA already on RISC. However, it should strand-invade the pri-miRNA and prevent cleavage by Dicer, so the pri-miRNA won't mature.
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com




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