changed over 1.5-fold in old compared to that of YA hearts. Among them, 34 miRNAs were up-regulated, while 31 were down-regulated (Table 1 and Figure 3, 4). 2. Multiple microRNA clusters are affected in the aging heart The miRNA genes are distributed across chromosomes either individually, or in clusters. A miRNA cluster is a group of miRNA genes located within a short distance on a chromosome. Based on miRBase database (http://www.mirbase.org) definition, clustered miRNAs are a group of miRNA genes located within 10 Kb of distance on the same chromosome. To test the hypothesis that adult aging may affect both individually distributed miRNAs and the microRNAs in clusters, we examine the genomic location of the 65 miRNAs that were differentially expressed in the aged heart. We found that approximately one half of the differentially expressed miRNAs was individually distributed and the other half was in miRNA clusters. The 32 miRNAs shown in Table 2 had 1.5-fold change in expression in old compared to that of YA hearts. The miRNA genes that corresponded to the 32 miRNA mature forms were in 11 clusters with their other cluster member(s) (Figure 5, 6, 7 and 8). Each clusters had at least 2 genes, with mir-466,467,669 cluster (cluster # 2) having 71 genes and mir-379,410 cluster (cluster #7) having 38 genes. Since genes in a miRNA cluster may share a common gene promoter and may be regulated as a whole transcriptional unit, it could be of interest to determine whether any entire cluster changed in the same direction, either up or down. After examination, it was found that the majority of the miRNAs in 10 of the 11 clusters (all clusters except cluster #7, Figure 6C) tended to change in the same direction as the other members in that cluster (Figure 5, 6 and 7), although not all of them had a 1.5-fold change (p,0.01) in expression, and not every change was significant (p.0.05). 3. Age-related changes in the expression of miRNA guide strands and their corresponding primary transcripts The transcription of pri-miRNA is the first step in miRNA biogenesis [32]. Pri-miRNA is first cleaved by the microprocessor complex into pre-miRNA, which is then cleaved by the RNase III enzyme Dicer and become mature microRNAs as a guide strand (miR) and a passenger strand (miR*). During typical miRNA biogenesis, the miR strand is preferentially selected for entry into a RISC complex, whereas the miR* strand could be degraded [33–35]. To test the hypothesis that transcriptional regulation plays an important role in the regulation of the mature microRNA form, we examined pri-miRNA and miR with real-time PCR. As shown in Figure 9, the miRNA guide strand miR-17, miR-21, miR-27a and miR-93 had an age-related increase accordingly with their corresponding primary transcripts pri-mir-17, pri-mir-21, pri-mir-27 and pri-mir-93. Similarly, both mir-466d primary transcript and miR-466d-3p showed an age-related decrease in expression. To further examine whether the expression of both pri-miRNA transcript and the miR strand change during maturation and aging, we compared the pri-mir-21 and the miR-21 levels in the mouse heart from 1 month to 24 months of age. As displayed in Figure 10, both miR-21 and pri-mir-21 levels showed no significant change from 1 month to 6 months of age, but started to increase after 6 months of age. Both miRNA forms reached their highest levels at the age of 18 months, then decreased afterwards; both curves showed a reversed ‘‘V’’ shape during adult aging. MicroRNA and microRNA Clusters in the Aging Heart Figure 7. The miRNA gene clusters and their genomic structures of cluster #7,cluster #11, which are part of 11 miRNA gene clusters shown in figure 5, 6 and 7. The number of miRNA genes in each cluster ranged from 2 to 71. In 10 out of 11 clusters, the expression of most miRNA genes in that cluster was in the same direction (the arrow q for increased expression, and the arrow Q for decreased expression). Please note that part of the miRNAs with changes in expression greater than 1.5 fold in old (O) vs. young adult (YA) hearts were listed in table 2. doi:10.1371/journal.pone.0034688.g007 4. Age-related changes in the expression of miRNA passenger strand (miR*) in the mouse heart In the cytoplasm, the pre-miRNA becomes cleaved by Dicer, which results in the formation of a 22 nt double-stranded miRNA duplex, with one being the guide strand (miR) and the other, the passenger strand (miR*). Recent deep-sequencing efforts indicate that a large number of miR*s are not degraded, but rather associate with Ago1 or Ago2 and remain functional [15,17–20]. An age-related change in the expression of miRNA passenger strands was observed in the present study. Five miR*s: miR-21*, miR-24-2*, miR-138*, miR-297a*, miR-467b* and miR-467e*, were decreased at least 1.5-fold, while miR-24-2* was increased at least 1.5-fold in old versus YA hearts (Figure 11). Interestingly, the fold of change in expression of miR-21* was the highest among all 65 miRNAs that were differentially expressed in old versus YA hearts (Figure 4 and 11). The level of miR-21* strand was examined in comparison with miR-21 and pri-mir-21 transcript from 1 month to 24 months of age. As shown in Figure 12, the miR-21* expression level did not PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34688