testis [49]). The remaining, non-annotated reads were then mined for characteristics of novel miRNAs using miRDeep.pl, using default settings [21]: these characteristics include the presence of reads corresponding to typical products of miRNA biogenesis; stability of the putative pre-miRNA hairpin; and homology to previously identified miRNAs. We considered miRNAs conserved if at least 50% of the overall sequence is identical and all of the seed sequence (nucleotides 2–7/8) matched separately. Results were individually confirmed by BLAST. MiRNA Target Prediction and Functional Analysis Targets for known and novel miRNAs were predicted using miRanda and TargetScan 5.0 software [23,24] against 39 UTR sequences obtained from the Ensembl and UCSC databases. Four separate predictions were conducted with different combinations of prediction software and 39 UTR sets (TS5.0 with Ensembl- derived 39 UTR, miRanda with Ensembl-derived 39 UTR, TS5.0 with UCSC-derived 39 UTR, and miRanda with UCSC-derived 39 UTR). Only common predictions were considered. Because TargetScan and miRanda predicted different numbers of target sites, the more conservative number was taken as the reported number of predicted target sites for a given miRNA–mRNA pair; these numbers were later used to rank target predictions. Gene annotations were retrieved using BioMart [50,51]. KEGG path- ways were retrieved using WebGestalt [52,53] for genes with more than 2 predicted miRNA target sites. P-value cut-off ,0.001 was chosen based on the recommendations in [54] for the application of a higher significance threshold for robust functional profiling of miRNA targets. P-values were corrected for multiple-hypothesis testing as part of the WebGestalt analysis using the Benjamini & Hochberg method [25]. MiRNA sequence alignment was conducted using Clustal W 2.0 [55,56]. Microarray expression profiling data of the following tissues in the brain of aging mice were retrieved from AGEMAP [28]: cerebrum, cerebellum, hippocampus, and striatum. Mice used in AGEMAP were C57BL/6 strain; in our study, we used C57BL/6J strain mice, a subline of C57BL/6. In order to compare with our dataset, only data for male mice aged 6- and 24-months old were analyzed; these time points correspond closely to the ages of mice used in our experiment. Percentile rank of expression change was calculated relative to the expression change of all 8932 genes assessed in each tissue. Statistical significance was determined by Welch’s two sample t-test (P-value ,0.05). Of the 43 insulin signaling pathway genes predicted to be targeted by down- regulated novel miRNAs, 11 were not found in the AGEMAP dataset. These genes are: Gck, Insr, Pklr, Prkaa2, Sh2b2, Eif4e1b, Pik3r5, Elk1, Ppp1r3a, Crkl, and Ptpn1. Quantitative Reverse Transcription PCR TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) were used according to manufacturer’s recommendations. MiRNA-specific primers for reverse transcription and qPCR were designed for nine novel miRNAs (all four upregulated miRNAs and top five downregulated miRNAs) (Figure 1(a)). Probes for snRNA U6, miR-124 and miR-155 were used as controls for the qRT-PCR. Expression levels relative to that of U6 were determined using the 2- DDCT method [57]. Statistical significance of expression change was determined by unpaired t-test. Supporting Information Figure S1 Insulin signaling pathway genes that are predicted to be targeted by novel miRNAs. Pathway retrieved from KEGG [26,27]. Boxes highlighted in red indicate Novel MicroRNAs in the Aging Mouse Brain genes that are predicted to be targeted by at least one novel miRNA. (PDF) Table S1 All 558 known mouse brain. (XLSX) miRNAs expressed in the Table S2 All known miRNAs expressed in the mouse brain that are differentially expressed (P-values ,0.05). Red: greater than 2.0-fold increase in old versus young mouse brains. Green: greater than 2.0-fold decrease in old versus young mouse brains. MiRNAs with read counts $10 in at least one time point sample indicated in bold. (XLSX) Table S3 MiRNA star sequences expressed in the mouse brain. Red: greater than 2.0-fold increase in old versus young mouse brains. Green: greater than 2.0-fold decrease in old versus young mouse brains. Differentially expressed miRNAs with P-value ,0.05 indicated in bold. (XLSX) Table S4 Novel miRNA candidates expressed in the mouse brain. MiRNA frequency was normalized by all reads that matched to the mouse genome (mm9) (Old/Young = 1.472107). {: candidates tested for by qRT-PCR. {: candidates with sequence overlap with known miRNAs (isomiRs). Blue font: miRNA new to mouse. Black font: completely novel miRNA sequence. * P-values for significance of differential expression calculated using DEGseq [20]. P-values ,0.05 indicated in bold. Three novel miRNA candidates overlap with snoRNA sequences (miR-6516, seq_692265_x97, seq_93483_x4) Two candidates mapped to multiple genomic regions including rRNA sequences when BLASTed. age: Ateles geoffroyi. bta: Bos taurus. dan: Drosophila ananassae. der: Drosophila erecta. dgr: Drosophila grimshawi. dme: Drosophila melanogaster. dmo: Drosophila mojavensis. dpe: Drosophila persimilis. dps: Drosophila pseudoobscura. dse: Drosophila sechellia. dsi: Drosophila simulans. dvi: Drosophila virilis. dwi: Drosophila willistoni. dya: Drosophila yakuba. gga: Gallus gallus. ggo: Gorilla gorilla. hsa: Homo sapiens. lla: Lagothrix lagotricha. mdo: Monodelphis domestica. mml: Macaca mulatta. mne: Macaca nemestrina. oan: Ornithorhynchus anatinus. ppa: Pan paniscus. ppy: Pongo pygmaeus. ptr: Pan troglodytes. sla: Saguinus labiatus. sme: Schmidtea mediterranea. xtr: Xenopus tropicalis. (XLSX) Table S5 Genes targeted by upregulated known miR- NAs ($2 target sites) and KEGG pathways (P-value .0.001) represented by those target genes. (XLSX) Table S6 Genes and pathways targeted by up- and downregulated novel miRNA candidates. (a) Genes targeted by upregulated novel miRNA candidates ($2 target sites) and KEGG pathways represented by those target genes (P-value .0.001). (b) Genes targeted by downregulated novel miRNA candidates ($2 target sites) and KEGG pathways represented by those target genes (P-value .0.001). (XLSX) Table S7 KEGG pathways targeted by multiple novel miRNA candidates (upregulated novel miRNAs or down- regulated novel miRNAs, collectively). (P-value .0.001). (XLSX) Table S8 Expression data of insulin signaling pathway genes retrieved from AGEMAP [28]. Expression levels of insulin signaling genes in four tissues of the brain were compared PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40028