Gold Standard 3 Sensitivity (%) Specificity (%) Youden Index number old criteria 1 + 2 45 26 2 1(FN) 8(TN) new criteria 2 + 39(TP) 7(FP) 86.7 73.1 ** 0.597 2 6(FN) 19(TN) miRNA miR-1 mir-124a mir-373 mir7 mir9 mir122a mir128a mir132 mir133a Gene Number (1) D/U 252/86 419/262 234/197 141/18 79/10 138/36 139/15 215/26 456/264 D/U 385947/152949 585637/459091 343051/327288 268092/26935 126313/10437 248250/44657 244848/28945 365154/29761 772975/306917 Total Bases (2) MRE(NL) (3) MRE(L) (4) Bases/MRE(NL) (5) Bases/MRE (L) (6) Ratio (7) D/U 267/49 787/330 392/237 245/14 138/5 325/44 263/15 239/10 742/204 D/U 752/1429 309/505 385/553 391/528 471/948 369/465 443/689 663/1062 509/655 D/U 1445/3121 744/1391 875/1380 1094/1923 915/2087 763/1014 930/1929 1527/2976 1041/1504 NL/L* 1.90/2.16 2.40/2.75 2.27/2.49 2.80/3.64 1.94/2.20 2.07/2.18 2.10/2.80 2.30/2.80 2.05/2.29 MicroRNA Central Loops Table 1. Statistical analyses of the results in MRE prediction before and after filtering with the central loop score TP: true positive; FP: false positive; FN: false negative; TN: true negative. Sensitivity~ TPTzPFN; Specificity~ FPTzNTN 1.Old criteria: seed homology plus minimum free energy ,215 kcal/mol, not filtered by the central loop score. Detailed description can be found in the Methods section. 2.New criteria: seed homology plus free energy ,215 kcal/mol, filtered by the central loop score. 3.Gold Standard: Luciferase activity assay. 4.**: The specificity comparison between old criteria and new criteria shows a significant difference (p,0.01, statistics analysis using McNemar’s Test). doi:10.1371/journal.pone.0001719.t001 According to the data in the Table 3, SD = [am+(1-a)n]TD, SU = [bm+(1-b)n]TU. The total number of bases in the 39-UTR in down-regulated genes is then assigned as BD and the total number of bases in up-regulated genes as BU. The ratio of bases per MRE-Up/ bases per MRE-D in the group screened for their loop scores (loop score. = 20) is bigger than that in a non-screened group (NL), so: BSDD ~ SUBD ~ ½amz(1 BSUU SDBU ½bmz(1 BD a)n TUBD w TBDU b)n TDBU TU amz(1 a)nwbmz(1 b)n (a b)(m n)w0 a.b, therefore m.n, meaning the ratio of MREs with loop scores . = 20 amongst true MREs is bigger than that in false MREs, so using the loop score as a criterion for MRE prediction is reasonable. Because miRNA:mRNA duplexes with type I decentered loops usually have a slightly shorter seed region with 6–7 nucleotides, it may be argued that the decreased repressive efficiency of miRNAs with type I decentered loops is the result of their short seed regions causing a decreased binding affinity, rather than the location of the central loop. To address this issue, we compared the repressive effects of two groups of miRNAs on gene expression in Table 4. The miRNAs in both groups have a short seed region of 6–7 nucleotides, with group 1 having standard loops and group 2 with type I decentered loops. The results demonstrate that the repressive efficiency of group 1 is much stronger than group 2. These results suggest that loop location indeed affect the efficiency of miRNA regulation. Table 2. Re-analysis of Lim et al and Grimson et al. microarray data from miRNAs transfected in HeLa cells. + 44(TP) 18(FP) 97.8 30.8 0.286 D/U 513 /107 1891/909 889/591 685/51 268/11 672/96 552/42 550/28 1518/468 Gene Number: The number of genes down-regulated or up-regulated by miR-1, miR-124a, or miR-373, with p,0.05 at both 12 hr and 24 hr. D: down-regulation. U: up-regulation. Total Bases: The total number of bases from the 39-UTR of the seed-containing mRNAs, which were either up- or down-regulated by miR-1, miR-124a, or miR-373. MRE (NL) and Bases/MRE(NL): MRE (NL) indicates the MREs scanned by FindTar version-1 with an MRE criteria of seed homologies and free energy ,215, but without a loop score. Bases/MRE(NL) = Total Bases/Total MRE (NL). MRE (L) and Bases/MRE (L): MRE (L) indicates MREs scanned by FindTar version-2 with an MRE criteria of seed homologies, free energy ,215, and loop score . = 20. Bases/MRE (L) = Total Bases/Total MRE (L). Ratio: Ratio = Bases per MREup/Bases per MREdown. Ratio (NL) = (5)U/(5)D; Ratio (L) = (6)U/(6)D. *: The ratio (NL) is significantly less than ratio (L) (p,0.05). doi:10.1371/journal.pone.0001719.t002 PLoS ONE | www.plosone.org 8 March 2008 | Volume 3 | Issue 3 | e1719