MicroRNA and microRNA Clusters in the Aging Heart Figure 4. The miRNAs that were decreased during adult aging. Thirty-one miRNAs were decreased in old (O) vs. young adult (YA) hearts. doi:10.1371/journal.pone.0034688.g004 The cardiac muscle cell line H9C2 was cultured in DMEM medium containing 10% newborn bovine serum (Invitrogen). Transient transfections were carried out with the Lipofectamine 2000 reagents (Invirogen) as previously described [24]. At 4 h after the transfection was initiated, the H9C2 cells were placed in Dulbecco’s modified Eagle’s medium with 10% newborn bovine serum and incubated overnight. The cells were then harvested and the total RNA was isolated using miRNeasy Mini kit (Qiagen) and Table 2. The 32 miRNAs and their corresponding miRNA gene clusters. Cluster # #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 Total: 11 miRNA clusters miRNA cluster mir-29b-2,29c mir-466-467-669 mir-106b-93-25 mir-29b-1,29a mir-290,293,295 miR-23a,27a,24-2 mir-379,411,758 mir-23b-27b-24-1 mir-17-19a-92a-1 mir-106a-92a-2 mir-222,221 miRNAs differentially expressed in O vs YA hearts miR-29c miR-466a-5p, miR-466b-5p, miR-466c-5p miR-297a*, miR-466d-3p, miR-466d-5p, miR-466f-3p, miR-466i, miR-467b*, miR-467e*, miR-467f, miR-467f, miR-467g,miR-669d, miR-669e, miR-669f miR-290-5p, miR-291b-5p miR-106a miR-29b miR-27a miR-380-5p miR-27b miR-25, miR-93, miR-221, miR-222 32 miRNAs miR-17, miR-19a, miR-19b, miR-20a Approximately half of the miRNAs (32 out of 65 miRNAs) that were differentially expressed in the old heart belong to 11 miRNA gene clusters. These 32 miRNAs had greater than 1.5-fold change in old (O) versus young adult (YA) hearts. Other miRNAs in the clusters with less than 1.5-fold change in expression are not shown in this table. doi:10.1371/journal.pone.0034688.t002 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34688