Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 Table 1 List of miRNAs conserved among various bilaterian animals miRNA family let-7 miR-1 miR-124 miR-125/lin-4 miR-34 H. sapiens let-7a miR-1 miR-124 miR-125b miR-34a Species M. musculus let-7a miR-1 miR-124 miR-125b-5p miR-34a G. gallus let-7a miR-1a miR-124a miR-125b miR-34a optimal free-energy information with a requirement of complete seed-sequence-matching using the RNAhybrid software [15], which predicts potential binding sites of short RNAs among target sequences. For this purpose, the optimal free energy was determined beforehand to efficiently cover experimentally validated miRNA/target- gene pairs. We calculated the free energy of 139 experi- mentally validated miRNA/target-gene pairs with a com- plete match of seed sequence, as assessed using RNAhybrid; subsequently, we defined the optimal free energy as < -17 kcal/mole of potential miRNA/target- gene pairs (data not shown). The evaluation of target- gene extraction during each step was carried out using an index termed “Enrichment” (see Methods section). As a result, in step 1, the number of potential target- gene candidates decreased from 357,430 to 153,387, with an enrichment index of 1.8. This prediction set contained 112 of the 145 experimentally validated tar- get-gene pairs. In step 2, we considered the 3’-UTR binding pattern of the miRNA/target mRNA. We defined four binding parameters (i.e., number of mismatches of mRNA within the whole miRNA sequence, number of mismatches of miRNA within the whole miRNA sequence, number of G-U wobble pairs within the whole miRNA sequence, and number of G-U wobble pairs within the seed sequence) of the hybridization pattern (Additional file 1) for optimisation of the thresholds for each of the fea- tures used to predict reliable miRNA/mRNA pairs. The Page 3 of 13 95.5 90.9 82.6 81.8 79.2 D. melanogaster C. elegans Identity (%) let-7 miR-1 miR-124 miR-125 miR-34 let-7 miR-1 miR-124 lin-4 miR-34 ranges of the four binding parameters were determined by calculating the coverage of 112 miRNA-mRNA pairs verified experimentally and of 153,387 miRNA-mRNA pairs predicted for each binding feature (Figure 2A-D). Five hundred parameter combinations were plotted on a 2D graph, using “Enrichment” on the X-axis and “Ratio of experimentally verified miRNA/mRNA” on the Y-axis (Figure 2E). From these parameter combinations, we defined the optimal combination of binding parameters for efficient screening based on a maximum EC value of 184.7 (number of mismatches of mRNA within the whole miRNA sequence: 12; number of mismatches of miRNA within the whole miRNA sequence: 10; number of G-U wobble pairs within the whole miRNA sequence: 4; and number of G-U wobble pairs within the seed sequence: 0). Accordingly, the number of potential tar- get-gene candidates was reduced from 153,387 to 28,201 and the experimentally validated target genes decreased from 112 to 76 after introduction of the criterion of optimal hybridization pattern (Enrichment index, 6.6) (Table 2). Finally, in step 3, we incorporated orthologous gene information and extracted genes that were evolutionarily conserved among more than four diverse bilaterian ani- mals, including H. sapiens. As a result, the number of predicted miRNA/target-gene pairs was minimized sub- stantially, from 10,356 to 31, using a significantly high Enrichment index of 25.7 (Table 2). The number of pre- dicted miRNA/target-gene pairs was especially high for Ratio of predicted miRNA/target-gene pairs (%) 357430/357430 (100) 153387/357430 (42.9) 28201/357430 (7.9) 31/10356* (0.3) Ratio of experimentally verified miRNA/target-gene pairs (%) 145/145 (100) 112/145 (77.2) 76/145 (52.4) 4/52 (7.7) Enrichment 1.0 1.8 6.6 25.7 Table 2 Summary of target gene extraction after each screening step H. M. G. D. C. sapiens musculus gallus melanogaster elegans All possible miRNA/gene pairs STEP 1 (Free energy and seed region) STEP 2 (Optimal parameter) STEP 3 97820 56666 98790 53420 12405 11256 53995 20410 2524 31 52850 14098 1118 53975 8793 898 The total number of possible target genes for the five miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) is represented for the five model species at each screening step (see Figure 1). Asterisks indicate the number of orthologous genes conserved among at least four species. Enrichment was calculated as the “ratio of experimentally verified miRNA/target-gene pairs” divided by the “ratio of predicted miRNA/target-gene pairs”.