The average ratio (n = 8) of acasp to [beta] actin transcript quantity was 0.17 in untreated controls compared to 0.51 in DMRIE-C controls.
RNAi using acasp dsRNA was effective at knocking down caspase activity, as indicated by a significant 30% decrease in activity, to 31.1 [+ or -] 1.1 FU x mg protei[n.sup.-1], in acasp dsRNA-incubated animals.
Specifically, dsRNA synthesized from acasp, a caspase with homology to vertebrate apoptotic executioner caspase 3 (Dunn et al., 2006), achieved RNAi of caspase 3 activity, using an optimized chemical transfection delivery.
During optimization of dsRNA, initial trials of both acasp and [beta] actin dsRNA showed no significant change in phenotype or successful knockdown at the lowest concentrations, but deleterious phenotypic effects--including cell death, tissue degradation, and animal mortality--were seen at the highest concentrations.
The template lengths for acasp and [beta] actin dsRNAs were designed in accordance with other invertebrate studies.
Acasp was more abundant in endodermal than ectodermal cells.
Successful gene knockdown with RNAi was also demonstrated with the caspase 3 activity assay, which showed the gene-specific phenotypic knockdown of acasp. Anemones treated with acasp dsRNA had significantly less caspase 3 activity than controls.
Delivery of acasp dsRNA did not achieve 100% knockdown; such failures are common in initial transfection.
The use of acasp RNAi will also enable us to answer key questions about the roles of cnidarian apoptosis in the onset and breakdown of symbiosis.