Abstract
The nematode Caenorhabditis elegans is often employed in investigations of diverse aspects of biology, including behavior, development, basic cellular processes, and disease states. The ability to utilize double-stranded RNA (dsRNA) to inhibit specific gene function in this organism has dramatically increased its value for these kinds of studies and has provided more flexibility in experimental design that include procedures. Here, we have collected a set of protocols from the C. elegans community for propagation of C. elegans, techniques for dsRNA preparation, four basic methods for delivery of dsRNA to C. elegans (injection, soaking, feeding, and in vivo delivery), and we suggest schemes that should facilitate detection of specific gene silencing.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Hubbard, E. J. and Greenstein, D. (2000) The Caenorhabditis elegans gonad: a test tube for cell and developmental biology. Dev. Dyn. 218, 2–22.
Timmons, L., Court, D. L., and Fire, A. (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112.
Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., and Plasterk, R. H. (2002) Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr. Biol. 12, 1317–1319.
Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons, L., Plasterk, R. H., and Fire, A. (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71–94.
Stiernagle, T. (1999) Maintenance of C. elegans, in C. elegans: A Practical Approach (Hope, I. A., ed.), Oxford University Press, Oxford, pp. 51–67.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Gonczy, P., Echeverri, C., Oegema, K., et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336.
Dudley, N. R., Labbe, J. C., and Goldstein, B. (2002) Using RNA interference to identify genes required for RNA interference. Proc. Natl. Acad. Sci. USA 99, 4191–4196.
Grishok, A., Tabara, H., and Mello, C. C. (2000) Genetic requirements for inheritance of RNAi in C. elegans. Science 287, 2494–2497.
Jin, Y. (1999) Transformation, in C. elegans: A Practical Approach (Hope, I. A., ed.), Oxford University Press, Oxford, pp. 69–96.
Epstein, H. F. and Shakes, D. C. (1995) Caenorhabditis elegans: Modern Biological Analysis of an Organism, vol. 48. Academic, San Diego.
Kimble, J., Hodgkin, J., Smith, T., and Smith, J. (1982) Suppression of an amber mutation by microinjection of suppressor tRNA in C. elegans. Nature 299, 456–458.
Stinchcomb, D. T., Shaw, J. E., Carr, S. H., and Hirsh, D. (1985) Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484–3496.
Tabara, H., Grishok, A., and Mello, C. C. (1998) RNAi in C. elegans: soaking in the genome sequence. Science 282, 430–431.
Maeda, I., Kohara, Y., Yamamoto, M., and Sugimoto, A. (2001) Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11, 171–176.
Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and Mello, C. C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.
Jorgensen, E. D., Durbin, R. K., Risman, S. S., and McAllister, W. T. (1991) Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters. J. Biol. Chem. 266, 645–651.
Logel, J., Dill, D., and Leonard, S. (1992) Synthesis of cRNA probes from PCR-generated DNA. Biotechniques 13, 604–610.
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., and Ahringer, J. (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, RESEAR CH0002.
Williams, B. D., Schrank, B., Huynh, C., Shownkeen, R., and Waterston, R. H. (1992) A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics 131, 609–624.
Mello, C. C., Kramer, J. M., Stinchcomb, D., and Ambros, V. (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Hull, D., Timmons, L. (2004). Methods for Delivery of Double-Stranded RNA into Caenorhabditis elegans . In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:023
Download citation
DOI: https://doi.org/10.1385/1-59259-775-0:023
Publisher Name: Humana Press
Print ISBN: 978-1-58829-242-1
Online ISBN: 978-1-59259-775-8
eBook Packages: Springer Protocols