Quality control parameters for RNAi reagents at different stages of the design pipeline. (a) Long dsRNAs that have regions of low complexity, for example, CA[ATCG] repeats or simple nucleotide repeats, can exert unspecific and cytotoxic effects. The quality of the primer designs used to synthesize amplicons from DNA sources is crucial, in particular when the synthesis is performed in 96- or 384-well formats where primers should have similar melting temperatures. (b) Dicer-mediated cleavage of long dsRNAs leads to the generation of siRNAs of lengths between 19 and 23 nucleotides . The quality of siRNAs depends on their ability to efficiently enter the RNA-induced silencing complex (RISC) and to access the target mRNA. This is influenced by thermodynamic properties, base preferences and chemical modifications. The specificity of siRNAs is influenced by sequence-independent and sequence-dependent features. siRNAs can trigger interferon responses or show concentration-dependent cytotoxic effects, independent of their sequence. Silencing of unintended target transcripts can occur through perfect and imperfect sequence homologies to the siRNA and through 'seed matches' to the transcript 3' UTRs. See text for details.
Horn et al. Genome Biology 2010 11:R61 doi:10.1186/gb-2010-11-6-r61