Overall flow of siRNA selection in siDirect 2.0. The functional and target-specific siRNAs were selected by three selection steps. In Step 1, functional siRNA sequences were selected according to our algorithm . In Step 2, siRNAs with Tm values below 21.5°C in the seed-target duplex were selected. In Step 3, nucleotides positioned in the 2-20 of both strands of the siRNAs were subjected to the near-perfect match searches, and siRNAs that have at least two mismatches to any other non-targeted transcripts were selected. The percentages denote the proportions of selected ('Yes') or unselected ('No') siRNA candidates calculated using all 23-mer subsequences (56,375,087; 100%) generated from human mRNAs in RefSeq release 30.