How much Morpholino to inject into a zebrafish egg or early zygote?

An injection volume of 500 pL is reasonable (Rosen et al. 2009, http://www.jove.com/video/1115/microinjection-of-zebrafish-embryos-to-an...). The basic rule for dosing of Morpholino injections is to use the least dose that gives you the phenotype of interest. As the dose increases, so does the potential for off-target RNA interactions and, with that, the potential of seeing a phenotype that has nothing to do with your target RNA. The higher the dose of oligo, the more mispairs will be tolerated in RNA binding. The probability of an off-target interaction increases with increasing oligo CG content and less strongly with increasing oligo length, affecting the "safe" dose, but we have no calculation to address this. Exceeding about 5 ng per embryo increases the risk of seeing a phenotype caused by an off-target interaction; using lower doses, you can have more confidence that the phenotype is due to interaction with the target RNA though specificity controls should still be performed, while at higher doses you need to be be increasingly rigorous about your specificity controls (I'd prefer to see doses stated by mole, but there is a strong tradition of writing doses by mass). However, you shouldn't trust that controlling dosing will eliminate off-target effects; there are two other steps that increase confidence that the observed phenotype is due to interaction with the target RNA and not an unexpected RNA. First, coinject a p53 oligo to prevent phenotypic changes triggered by loss of the protein of interest and caused by p53-mediated apoptosis (See Robu et al. 2007, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1877875/). Second, use a nonoverlapping Morpholino targeting the same RNA as a specificity control. If the two oligos produce the same phenotype when separately injected, this supports the hypothesis that the phenotype is due to knockdown of the targeted RNA and not an interaction with an unexpected RNA. If the oligos elicit the phenotype with dose synergy when coinjected, this further supports specificity of the phenotype (Bill et al. 2009, http://online.liebertpub.com/doi/pdfplus/10.1089/zeb.2008.0555).

I'd appreciate any feedback on this discussion. Thanks!