A protocol for designing siRNAs with high functionality and specificity

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.