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.
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.
Authors: David J Forsthoefel; Noëlle P James; David J Escobar; Joel M Stary; Ana P Vieira; Forrest A Waters; Phillip A Newmark Journal: Dev Cell Date: 2012-10-16 Impact factor: 12.270
Authors: T Harshani Peiris; Daniel Ramirez; Paul G Barghouth; Udokanma Ofoha; Devon Davidian; Frank Weckerle; Néstor J Oviedo Journal: Development Date: 2016-03-24 Impact factor: 6.868
Authors: Prasad Abnave; Ellen Aboukhatwa; Nobuyoshi Kosaka; James Thompson; Mark A Hill; A Aziz Aboobaker Journal: Development Date: 2017-09-11 Impact factor: 6.868
Authors: Christopher P Arnold; M Shane Merryman; Aleishia Harris-Arnold; Sean A McKinney; Chris W Seidel; Sydney Loethen; Kylie N Proctor; Longhua Guo; Alejandro Sánchez Alvarado Journal: Elife Date: 2016-07-21 Impact factor: 8.140