UNIT 12.3 RNAi: Design and Analysis RNAi, or RNA interference, is the specific silencing of a gene by a double-stranded RNA (dsRNA) comprising a strand homologous to the gene (see Background Information). The RNAi pathway is conserved across species (all higher eukaryotes have it, but, in yeast, S. cerevisiae seems to have lost the pathway, while S. pombe seems to have portions of it). The RNAi pathway is implicated in diverse processes, such as gene silencing (either through mRNA degradation or blocking mRNA translation), chromatin maintenance, centromere silencing, epigenetic control, and genomic stability (Hannon, 2002; Denli and Hannon, 2003; Bartel, 2004). The study and use of RNAi requires bioinformatics broadly in six main areas: 1. Study of the RNAi process and proteins involved in it, which involves searching for motifs using HMMs and phylogenetic analysis. This can help uncover the mechanism of RNAi, in turn helping with better silencing designs (see Background Information). 2. Prediction of novel miRNA genes. Discovery of new miRNA genes can help iden- tify critical features that control the silencing behavior of dsRNAs. MiRscan and MiRseeker are two programs that have been used to identify novel miRNA genes (see Basic Protocol 3). 3. Identification of miRNA targets and understanding the temporal and spatial modulation of miRNA expression (see Basic Protocol 4). Understanding targeting of miRNAs would provide an understanding of their biological function and help in more precise designs of siRNA and shRNAs. 4. Design of oligos for silencing (siRNA, see Basic Protocol 1; and shRNA, see Basic Protocol 2). This incorporates knowledge gained from the studies listed above. 5. Building a genome-wide RNAi silencing library (see Alternate Protocol 1). 6. Use of the library in functional screens (see Alternate Protocol 1). The complexity and size of the RNAi library makes bioinformatics an indispensable tool in designing studies and interpreting results. BASIC PROTOCOL 1 DESIGNING siRNAs siRNAs are now routinely used to silence genes in vitro. They can be ordered off-the-shelf for common genes, and a lot of effort has gone into understanding their action. Given here are the procedures that can help design siRNAs that are likely to be functional. The disadvantages of the use of siRNAs include the fact that siRNAs are expensive and that the use of high dosage (each cell usually receives several siRNAs) makes induction of other effects more likely. The advantages of this method, on the other hand, are that one can usually find well char- acterized siRNAs for several genes in the literature. Several companies (e.g., Dharmacon and Ambion; see Internet Resources) sell well characterized off-the-shelf siRNAs for genes, which take the guesswork out of experiments. The steps of this procedure have been implemented on the Web server at (http://katahdin.cshl.org:9331/siRNA). Dharmacon has a Web site (see Internet Re- sources) that allows design of siRNAs. Contributed by Ravi Sachidanandam Current Protocols in Bioinformatics (2004) 12.3.1-12.3.10 Copyright C  2004 by John Wiley & Sons, Inc. Analyzing RNA Sequence and Structure 12.3.1 Supplement 6