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RNAi: Design and Analysis

Ravi Sachidanandam¹

¹Sachidanandam Lab, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Publication Name:

Current Protocols in Bioinformatics

Unit Number:

Unit 12.3

DOI:

10.1002/0471250953.bi1203s6

Online Posting Date:

September, 2004

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Abstract

RNAi is a novel genetic tool that is poised to revolutionize the study of genetics and gene networks. Use of this tool requires knowledge of the underlying mechanisms and application of this knowledge to the construction of a gene-silencing library. This unit discusses approaches to rational design of this library and its use in experiments. It focuses on the design algorithms developed so far and briefly discuss several issues that could lead to improvements in the current design algorithms.

Keywords: RNAi; Oligo design; siRNA; miRNA; shRNA

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Unit Introduction
Basic Protocol 1: Designing siRNAs
Support Protocol 1: Constructing a Database of Nonredundant mRNA Sequences
Basic Protocol 2: shRNA Design
Basic Protocol 3: Prediction of miRNA Genes
Basic Protocol 4: Identifying Targets of miRNA
Alternate Protocol 1: Building and Using a Library of shRNAs
Commentary
Literature Cited
Figures
Tables

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Figures

Figure 12.3.1

The design of hairpins.

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Figure 12.3.2

Output from mfold for the author's hairpin design.

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Figure 12.3.3

The processes involved in RNAi. Abbreviations: MNT, micrococcal nuclease tudor; FXR, fragile X–related; VIG, vasa intronic gene.

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Literature Cited

Literature Cited
	Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116:281-297.
	Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beijersbergen, R.L., and Bernards, R. 2004. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428:431-437.
	Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. 2002. The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16:2733-2742.
	Denli, A.M. and G.J. Hannon 2003. RNAi: An ever-growing puzzle. Trends Biochem. Sci. 28:196-201.
	Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
	Hannon, G.J. 2002. RNA interference. Nature 418:244-251.
	Hemann, M.T., Fridman, J.S., Zilfou, J.T., Hernando, E., Paddison, P.J., Cordon-Cardo, C., Hannon, G.J., and Lowe, S.W. 2003. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33:396-400.
	Khvorova, A., Reynolds, A., and Jayasena, S.D.. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209-216.
	Lee, J.B. and Warc, B. 2002. Open Source Web Development with LAMP: Using Linux, Apache, MySQL, Perl, and PHP. Addison-Wesley, Reading, Mass.
	Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin D.S. 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16:948-958.
	Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O'Shaughnessy, A., Gnoj, L., Scobie, K., Chang, K., Westbrook, T., Cleary, M., Sachidanandam, R., McCombie, W.R., Elledge, S.J., and Hannon, G.J. 2004. A resource for large-scale RNA-interference-based screens in mammals. Nature 428:427-431.
	Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., and Bartel, D.P. 2002. Prediction of plant microRNA targets. Cell 110:513-520.
	Schwartz, R.L. and Phoenix, T. 2001. Learning Perl. O'Reilly & Associates, Sebastopol, Calif.
	Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199-208.
Internet Resources
	http://www.ambion.com/catalog/supp/silencer_lib.html
Ambion company Web site.
	http://www.apache.org
Apache open source Web server.
	http://www.ebi.ac.uk/clustalw/
ClustalW: Open source multiple alignment program..
	http://katahdin.cshl.org:9331/siRNA
Cold Spring Harbor Laboratory siRNA Web site (R. Sachidanandam and J. Lee).
	http://www.dharmacon.com/
Dharmacon company Web site.
	http://design.dharmacon.com/rnadesign/default.aspx sid=731615029
Dharmacon siRNA Design Center.
	http://www.cshl.org/public/SCIENCE/hannon.html
Hannon Laboratory Web site at Cold Spring Harbor Laboratory.
	http://hmmer.wustl.edu/
HMMER Web site (S.R. Eddy). Open source HMM tools.
	http://www.bioinfo.rpi.edu/applications/mfold/
mfold: Open source RNA folding program (M. Zuker).
	http://genes.mit.edu/mirscan/
MiRscan Web site (M. Mawson, L.P. Lim, and N. Lau).
	http://toy.lbl.gov:9050/cgi-bin/miRseeker.pl
MiRseeker Web site (P. Tomancak).
	http://www.mysql.com
MySQL: Open source database.
	http://www.ncbi.nlm.nih.gov/
NCBI: A site for genomics data, tools, and analyses.
	http://igs-server.cnrs-mrs.fr/Tcoffee/
T-Coffee server.