Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 Page 5 of 13 Figure 2 Parameters used for the prediction of miRNA/mRNA pairs and their coverage. To optimize the binding parameters of miRNA/ mRNA duplexes, we determined the coverage of four binding parameters (mismatch of mRNA within the whole miRNA sequence (A), mismatch of miRNA within the whole miRNA sequence (B), G-U wobble pairs within the whole miRNA sequence (C), and G-U wobble pairs within the seed sequence (D)). Calculation of the coverage was performed using 112 experimentally verified miRNA/mRNA pairs (orange line) and 153,387 predicted miRNA/mRNA pairs (blue line). The yellow bar indicates differences in coverage between experimentally verified and computationally predicted miRNA/mRNA pairs. Four or five points chosen from the highest yellow bar were used as the range of each of the four binding parameters (blue squares) used in this study. We determined parameter space using the binding patterns of the miRNA/mRNA pairs based on four features (E). Five hundred parameter combinations were plotted on a 2D graph using “ratio of experimentally verified miRNA/mRNA” on the Y-axis and “Enrichment” on the X-axis. Black circles (57.3% of the coverage and 3.2-fold of the Enrichment) indicate the point that corresponded to optimized parameters for the prediction of final conserved miRNA/target pairs: 12 for the number of mismatches in the mRNA, 10 for the number of mismatches in the miRNA, 4 for the number of G-U wobble pairs within the whole miRNA sequence, and 0 for the number of G-U wobble pairs within the seed sequence (see Methods section).