miRNA gene knockdown? - (Nov/06/2007 )
I am carrying out antisense-mediated knockdown of a miRNA by an LNA oligo. I observed what might be success. On a northern from a non-denaturing gel, I observed that the band normally detectable at 23nt is no longer present in my experimental samples, and still detectable in the control. However, I observe a strong band at a higher position (est. 40nt) in my experimental lanes. I suspect that this may be double-stranded nucleic acid (LNA:RNA) of the antisense oligo binding up the miRNA, which might indicate that I was successful in knocking down the miRNA, if it is binding up the miRNA, preventing it from being utilizable.
Further evidence of this is that when I examine a northern where I run my samples on a denaturing gel, I instead see a band running at the same position as the control. The fact that it could be double stranded and still bind to a membrane is possible as I am using a (+) charged nylon membrane and not nitrocellulose.
My question is this... Has anyone else observed this phenomena? I have tried to look in the literature but don't see it. We considered that it could be pri-miRNA, but if so, why is it not a larger size? If degraded pri-miRNA, it would probably not be a distinct band, and also why the change between non-denaturing and denaturing gel?
Do you think that this is double stranded nucleic acid? If not, what do you think it might be?
Thank you for your help...
Your hypothesis that the bandshift is due to the LNA binding seems reasonable to me. What about using a labeled LNA for the knockdown? Then you would extract and run out the small RNA on the gel and see whether the labeled oligos, after some time in cells with miRNAs, have the same migration as the putative heteroduplex band you observed. If the mobility matches that would be good support for your hypothesis.