miRNAs, the RISC is thought to inhibit protein production either through blocking translation or by reducing messenger RNA stability [21,22]. Previously, the microRNA passenger strand (miR* or miRNA*) was thought to be degraded without significant biological impact on gene regulation. Recent studies, including deep-sequencing efforts, however, indicate that a large number of miR*s are not simply degraded, but rather, associate with Ago1 and/or Ago2 and are also capable of silencing target transcripts [15,16,18,19]. To determine the impact of advancing age on microRNA expression in the heart, we examined the microRNA expression level in old versus young adult (YA) hearts. We found that the expression of 65 microRNAs, including 11 miRNAs clusters was significantly altered during adult aging. In addition, we found that major changes in the expression of miRNA occurred from the post-maturational period through adult aging. Our data suggest that transcriptional regulation of microRNA transcripts and altered expression of Argonaut proteins contribute to age-related changes in the expression of both miR and miR* strands. Materials and Methods Animal tissues Healthy C57BL/6 mice were obtained from colonies main- tained by the National Institute of Aging (NIA), the National Institutes of Health, under contractual agreement with Harlan Sprague-Dawley, Inc. (Harlan, IN). After euthanasia, the hearts were removed from mice and subjected to standard RNA isolation and histological procedures. Some heart tissue samples (4 months and 24 months) were obtained from the Aged Rodent Tissue Bank at NIA. For each time point, there were three independent biological replicates. The studies were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at Central Arkansas Veterans Healthcare System (IACUC# 4-02-03) and in accordance with the NIH Guiding Principles for Research Involving Animals and Human Beings. MicroRNA and microRNA Clusters in the Aging Heart Total RNA isolation All RNA samples were first isolated from the mouse cardiac ventricles using UltraSpec RNA Isolation Reagent as previously described. To minimize mouse DNA contamination and enrich the small RNA fraction, the total RNA samples were purified using miRNeasy Mini kit (Qiagen) and RNase-free DNase I according to the manufacture’s instruction manual [23,24]. MicroRNA arrays The ventricular tissue samples used for the microRNA array analysis were obtained from healthy young adult (4-month-old) and old (24-month-old) C57BL6 mice. The RNA sample isolation and microRNA array were performed in triplets for young adult and old animals. A total of 6 RNA samples of ventricular tissue representing 3 individual young adult and 3 individual old mice were shipped on dry ice to Exiqon, Inc., which provided the service for RNA quality verification, microRNA array hybridiza- tion and comprehensive statistical analysis. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under ac- cession no. GSE32935. Briefly, each pair of young adult and old mouse samples were labeled with Hy3 and Hy5 fluorescent dyes respectively and hybridized to a miRCURY LNATM mouse microRNA Array (version 11.0), which held 648 mature micro- RNA probes, as well as perfectly matched and mismatched probes for quality control. After signal amplification, the background was subtracted and normalized using LOWESS (Locally Weighted Scatter plot Smoothing) regression algorithm. This within-slide normalization was performed to minimize differences between the colors in an intensity-dependent manner. The array output was received in Excel spreadsheets containing the normalized micro- RNA expression profiles in each heart sample; the expression comparison between old versus young adult heart samples and ‘‘Expression Matrix’’ containing normalized Hy3/Hy5 ratios (log2 transformed) from all hybridizations. The list was sorted based on the most variant expressed miRNAs comparing the two sample types. 65 miRNAs passed the filtering criteria with an average ‘‘log-Median-Ratio’’ .0.58, which represents at least .1.5-fold change in microRNA expression, and the microRNA expression in all three pairs (young adult versus old) was in the same direction. Figure 1. The signal intensity of miRNAs expressed in the mouse heart. There was no significant difference in the overall signal intensity of total microRNAs between young adult (YA) and old hearts. doi:10.1371/journal.pone.0034688.g001 Figure 2. The number of miRNA that were differentially expressed in the heart. Among the 599 microRNAs that were expressed in the heart, 272 miRNAs were down-regulated, while 327 miRNAs were up-regulated in the old compared with young adult hearts. doi:10.1371/journal.pone.0034688.g002 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34688