RNA interference by feeding in vitro–synthesized double‐stranded RNA to planarians: Methodology and dynamics

• Published in: Developmental dynamics 2013-06, Vol.242 (6), p.718-730
• Main Authors: Rouhana, Labib, Weiss, Jennifer A., Forsthoefel, David J., Lee, Hayoung, King, Ryan S., Inoue, Takeshi, Shibata, Norito, Agata, Kiyokazu, Newmark, Phillip A.
• Format: Article
• Language: English
• Subjects: Animals; Bacteria - genetics; Developmental Biology - methods; Dugesia japonica; Gene Expression Regulation, Developmental; Genetic Techniques; Genetic Vectors; Phenotype; planarian; Planarians - genetics; Reproducibility of Results; RNA Interference; RNA, Double-Stranded - genetics; RNAi; Schmidtea mediterranea; siRNA
• ISSN: 1058-8388; 1097-0177
• DOI: 10.1002/dvdy.23950

Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double‐stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high‐throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718–730, 2013. © 2013 Wiley Periodicals, Inc. Key Findings In vitro‐synthesized dsRNA is fed to planarians to induce robust RNAi. Presentation of an improved protocol for RNAi in planarians amenable to high‐throughput studies. Analysis of dsRNA processing dynamics and spatiotemporal RNAi activity. Systematic comparisons of effects of variations in amount, frequency and mode of dsRNA delivery on RNAi efficiency.