Jon's Blog

This is about detecting the activity of a splice-modifying oligo by reverse-transcriptase PCR and gel electrophoresis.

The expected outcome of an exon targeting an exon 3 splice junction (either i2e3 or e3i3) is elimination of exon 3 from the mature mRNA (along with both of its flanking introns, i2 and i3). The typical way to assess activity of a splice-modifying oligo is to isolate RNA from treated samples and run reverse-transcriptase PCR (RT-PCR), then run the PCR product on a gel to assess the mass of the product against a DNA ladder.

Translational development of splice-modifying antisense oligomers.
Fletcher S, Bellgard MI, Price L, Akkari AP, Wilton SD.
Expert Opin Biol Ther. 2016 Nov 2:1-16. [Epub ahead of print]
http://www.tandfonline.com/doi/full/10.1080/14712598.2017.1250880

No Morpholino work in this one, but it explores a difference between targeting RNA versus DNA.

Cell-Intrinsic Adaptation Arising from Chronic Ablation of a Key Rho GTPase Regulator.
Cerikan B, Shaheen R, Colo GP, Gläßer C, Hata S, Knobeloch KP, Alkuraya FS, Fässler R, Schiebel E.
Dev Cell. 2016 Sep 28. pii: S1534-5807(16)30595-0. doi: 10.1016/j.devcel.2016.08.020.

http://www.sciencedirect.com/science/article/pii/S1534580716305950

Once again, a conversation with a new Morpholino user led to a discussion others might find useful.

For the two non-overlapping oligo specificity control, you do two separate sets of injections of the two oligos targeting the same miRNA. If the embryos treated with the oligo targeting the 5' end of the miRNA produces the same phenotype as the oligo targeting the 3' end of the miRNA, then that is good: it supports the idea that the phenotype you are seeing is caused by knocking down the activity of the miRNA you intend to target, and not caused by binding to an unexpected RNA.