Optimized protocols for RNA interference in Macrostomum lignano

Macrostomum lignano, a marine free-living flatworm, has emerged as a potent invertebrate model in developmental biology for studying stem cells, germline, and regeneration processes. In recent years, many tools have been developed to manipulate this worm and to facilitate genetic modification. RNA interference is currently the most accessible and direct technique to investigate gene functions. It is obtained by soaking worms in artificial seawater containing dsRNA targeting the gene of interest. Although easy to perform, the original protocol calls for daily exchange of dsRNA solutions, usually until phenotypes are observed, which is both time- and cost-consuming. In this work, we have evaluated alternative dsRNA delivery techniques, such as electroporation and osmotic shock, to facilitate the experiments with improved time and cost efficiency. During our investigation to optimize RNAi, we demonstrated that, in the absence of diatoms, regular single soaking in artificial seawater containing dsRNA directly produced in bacteria or synthesized in vitro is, in most cases, sufficient to induce a potent gene knockdown for several days with a single soaking step. Therefore, this new and highly simplified method allows a very significant reduction of dsRNA consumption and lab work. In addition, it enables performing experiments on a larger number of worms at minimal cost.