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Post by cassieconte » Mon Nov 28, 2011 5:49 pm

Hey all,

In miRNA:miRNA* duplexes, what exactly is the amiRNA*? I am currently reading a resesarch paper and am having some trouble understanding what they actually did in Figure 1 of this article (http://www.plantcell.org/content/18/5/1121.full). Why are primer pairs needed to replicate 1 single strand (i.e. primers III and IV for amiRNA*)?

Any clarifications would be appreciated. Thanks!

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Post by jonmoulton » Mon Nov 28, 2011 10:54 pm

In many miRNAs, the guide strand loads into the RISC and the star strand is degraded. That's the easy case, and you can unambiguously tell which strand is the guide strand. However, in some cases, in particular those for which both ends of the miRNA duplex have similar duplex stability, either the guide or the star strand can load into RISC, so you end up with a population of RISC that has some guide-directed activity and some star-directed activity. If an even amount of each strand is loaded onto RISC, the designation of which is the guide strand and which is the star strand is arbitrary.

As for the primer pairs, in the first round of RT-PCR only one primer is actually used to prime reverse transcriptase and a single strand of DNA is produced, complementary to the RNA template. In the second cycle of RT-PCR that new DNA strand binds to the other primer and another DNA strand is produced, complementary to the first (and the same as the RNA strand). Now you have both DNA strands, a complementary DNA duplex, and subsequent PCR cycles prime both of the strands, doubling the number of strands during each cycle.

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