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 complementarity 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 specific siRNAs.