TY - JOUR
T1 - RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians
T2 - Methodology and dynamics
AU - Rouhana, Labib
AU - Weiss, Jennifer A.
AU - Forsthoefel, David J.
AU - Lee, Hayoung
AU - King, Ryan S.
AU - Inoue, Takeshi
AU - Shibata, Norito
AU - Agata, Kiyokazu
AU - Newmark, Phillip A.
PY - 2013/6
Y1 - 2013/6
N2 - 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.
AB - 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.
KW - Dugesia japonica
KW - Planarian
KW - RNA-interference
KW - RNAi
KW - Schmidtea mediterranea
KW - SiRNA
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U2 - 10.1002/dvdy.23950
DO - 10.1002/dvdy.23950
M3 - Article
C2 - 23441014
AN - SCOPUS:84878137449
SN - 1058-8388
VL - 242
SP - 718
EP - 730
JO - Developmental Dynamics
JF - Developmental Dynamics
IS - 6
ER -