MicroRNA and microRNA Clusters in the Aging Heart Figure 5. The miRNA gene clusters and their genomic structures of cluster #1,cluster #4, 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.g005 RNase-free DNase I according to the manufacture’s instruction manual. Individual transfection experiments were carried out in triplicate, and the results were reported as average (mean 6 S.D.) from representative experiments. Statistical analysis Data are given as mean values 6 SD, with n denoting the number of experiments unless otherwise indicated. The differen- tially expressed microRNAs with at least a 1.5-fold change were identified using a t-test with a cut off p-value (p,0.05). Results 1. Differential expression of cardiac microRNAs in the aging heart It has been reported that microRNA expression was altered in cardiac hypertrophy, congestive heart failure and also in senescent cells [27–31]. To test the hypothesis that the expression of microRNA may also change during cardiac aging, we compared microRNA profiles in the young adult (YA, 4-month-old) versus Figure 6. The miRNA gene clusters and their genomic structures of cluster #5,cluster #8, 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). Only in one cluster, cluster #7 (Figure 6C), were most of the miRNA genes not expressed in the same direction. 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.g006 old (O, 24-month-od) mouse hearts. The Exiqon microRNA array (version 11.0) was employed for the microarray analysis. An overview of the total microRNA expression in YA versus old hearts showed that the total microRNA signal intensity was 311248 in YA hearts versus 310447 in old hearts (Figure 1); there was no significant difference between the two age groups (NS, n = 3). However, the comparison of each individual microRNA expression between old and YA showed that 45% of the microRNAs were down-regulated in old versus YA, while 55% of the microRNAs were up-regulated in old versus YA (Figure 2, p,0,01, n = 3). These data indicate that while the expression of many of the individual microRNAs changed in advancing age, the healthy old heart was still able to keep the overall microRNA expression close to that of the YA heart. To examine the miRNAs that were significantly altered during aging, the expression of miRNAs in YA versus old hearts were compared. It was found that the expression level of 65 miRNAs PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34688