Design, Manufacture, and Assay of the Efficacy of siRNAs for Gene Silencing

Small interfering RNAs (siRNAs) have been widely exploited for nucleotide-sequence-specific posttranscriptional gene silencing, as a tool to investigate gene function in eukaryotes, and they hold promise as potential therapeutic agents. Conventionally designed siRNAs are 21-mers with symmetric 2-nt 3′ overhangs that mimic intermediates (microRNAs or miRNAs) of the natural processing of longer dsRNA (double-stranded RNA). siRNAs are sequences with full c omplementarity to their target mRNA and can be generated by either chemical synthesis or processing of shRNAs (short hairpin RNAs) transcribed from DNA vectors. To minimize off-target effects, any homology to nontarget mRNA can be verified using the expressed sequence tag (EST) database for the relevant organism. Here, we provide a practical guide and an overview to the design and selection of effective and specifc siRNAs.