Table 1. The 65 miRNAs that were differentially expressed in old versus young adult hearts. Increased Decreased miR-15b, miR-17, miR-19a, miR-19b, miR-21*, miR-138*, miR-184, miR-20a, miR-21, miR-24-2*, miR-25, miR-290-5p, miR-291b-5p, miR-297a*, miR-27a, miR-27b, miR-29b, miR-29c, miR-325, miR-329, miR-34b-3p, miR-93, miR-106a, miR-142-3p miR-142-5p, miR-144, miR-146a, miR-146b,miR-186, miR-193, miR-199a-5p, miR-207, miR-221, miR-222, miR-223, miR-335-5p, miR-374, miR-380-5p, miR-425, miR-466a-5p, miR-466b-5p, miR-466c-5p, miR-466d-3p, miR-466d-5p, miR-466f-3p, miR-466i, miR-467b*, miR-467e*, miR-467f, miR-467g, miR-574-5p, miR-667, miR-669d, miR-669e, miR-669f, miR-1187, miR-1192, miR-1196 miR-455, miR-652, miR-668, miR-677 miR-693-5p, miR-706, miR-744, 34 miRNAs were increased, while 31 miRNAs were decreased in old heart. doi:10.1371/journal.pone.0034688.t001 Real-time RT-PCR quantitation of pri-miRNAs and mature miRNAs To quantitate the expression of the miRNA primary transcripts and the miRNA mature forms, Real-time RT-PCR was performed. To select a proper internal loading control for RT-PCR, we examined the expression of 5S ribosomal RNA (5S RNA) and U6 snRNA in young adult versus old hearts. We found that the expression of 5S RNA remained unchanged in young adult vs. old, while U6 expression changed significantly in young adult vs. old hearts. Therefore, 5S RNA was used as an internal loading control. Detection of pri-miRNAs. The primers for the detection of pri-miRNAs were designed using PRISM Primer Express 3.0 software (Applied Biosystems), and synthesized at Integrated DNA Technologies Inc. The first-strand cDNA synthesis was carried out MicroRNA and microRNA Clusters in the Aging Heart using random hexamer primer, and the PCR was performed using the following primers: pri-mir-17 forward 59-gctttggctttttcctttttg- 39, pri-mir-17 reverse 59-cctcactgcagtagatgcaca-39; pri-mir-21 forward 59-ccagagatgtttgctttgctt-39, pri-mir-21 reverse 59- tgccatgagattcaacagtca-39; pri-mir-27a forward 59- tttgatgcc agtcacaaatca-39, pri-mir-27a reverse 59-agccactgtgaacacgacttt-39; pri-mir-93 forward 59-cacctcacctaatgaccctca-39, pri-mir-93 reverse 59-caagtcctagccctcatggat-39; pri-mir-466d forward 59- cacatgcaa cacacacatatgaa-39 and pri-mir-466d reverse 59-ctgattctggcaa gcattttc-39. Detection of mature miRNAs. To detect the mature miRNA strand of either miR or miR*, the first-strand cDNA synthesis was carried out using a universal reverse primer and the Universal RT microRNA PCR system (Exiqon). The RT-PCR reagents and the primers for miR-17, miR-21, mir-21*, miR-27, miR-93, miR-466d-3p and the 5S RNA reference primers that were used as endogenous control were purchased from Exiqon. The PCR amplification was performed in a 7900HT Fast Sequence Detector System (Applied Biosystems) with following program: Cycle 1, 95uC for 10 minutes. Cycle 2, 40 cycles of 95uC for 15 seconds, 60uC for 60 seconds. Cycle 3, 95uC for 15 seconds, 60uC for 15 seconds, 95uC for 15 seconds. CT values were automatically obtained. Relative expression values were obtained by normalizing CT values of the miRNA genes in comparison with CT values of the endogenous control (5S RNA) using the CT method [25,26]. Transfection Assays The Ago1 expression plasmid is a gift of Dr. E. K. L. Chan, and the Ago2 expression plasmid is a gift of Dr. T. Tuschl. The plasmid construct that contains the mir-21 gene was generated by PCR and cloning. Briefly, the DNA fragment containing mir-21 gene was amplified using forward primer 59- tagaattc tgcccaggcttt- tatgtattg -39 and reverse primer 59- ta ctcgag ggcattgcttttcaagtatgg -39, and then cloned into pCDNA3.1(+) vector. The DNA construct was confirmed by sequencing analysis. Figure 3. The miRNAs that were increased during adult aging. Thirty-four miRNAs were increased in old (O) vs. young adult (YA) hearts. doi:10.1371/journal.pone.0034688.g003 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34688