RNA interference by feeding in vitro–synthesized double-stranded RNA to planarians: Methodology and dynamics
Article first published online: 1 APR 2013
Copyright © 2013 Wiley Periodicals, Inc.
Volume 242, Issue 6, pages 718–730, June 2013
How to Cite
Rouhana, L., Weiss, J. A., Forsthoefel, D. J., Lee, H., King, R. S., Inoue, T., Shibata, N., Agata, K. and Newmark, P. A. (2013), RNA interference by feeding in vitro–synthesized double-stranded RNA to planarians: Methodology and dynamics. Dev. Dyn., 242: 718–730. doi: 10.1002/dvdy.23950
- Issue published online: 20 MAY 2013
- Article first published online: 1 APR 2013
- Accepted manuscript online: 26 FEB 2013 12:00AM EST
- Manuscript Accepted: 14 FEB 2013
- Manuscript Revised: 13 FEB 2013
- Manuscript Received: 11 JAN 2013
- NSF. Grant Number: 0804021
- NIH. Grant Number: R01 HD043403
- Schmidtea mediterranea;
- Dugesia japonica
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.
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.
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.