miRNA expression atlas in male rat

Animal experiments

For each miRNA microarray experiment, 9-week-old male Sprague-Dawley rats were obtained from Charles River Japan, Inc. (Kanagawa, Japan). After a 7-day quarantine and acclimatization period, 10-week-old animals were used (N=6). The animals were individually housed in stainless-steel cages in an animal room set to the following conditions: 12 h (7:00–19:00) light phase; ventilation rate, 12/h; temperature, 20 °C–26 °C; and relative humidity, 35–75%. Each animal had free access to water and pellet diet (CRF-1, sterilized by radiation, Oriental Yeast Co., Ltd., Tokyo, Japan). Each animal was anesthetized with ether. The animals were divided into 2 groups to compare the effects of peripheral blood cells in organs; 3 animals were perfused with saline to remove blood from all organs, while the remaining 3 animals that were not perfused were subjected to collection of only the heart (atrium and interventricular septum), kidney, liver and lung. All organs evaluated in this study are listed in Table 1 (available online only). Experimental protocols were reviewed and approved by the Ethics Review Committee for Animal Experimentation of the National Institute of Health Sciences.

RNA extraction and miRNA microarray analysis

RNA was prepared using the miRNeasy kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was quantified using a DU-7400 spectrophotometer (Beckman Coulter, Brea, CA) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Cyanine-3 (Cy-3) labeled RNA was prepared from 0.1 μg RNA using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies), according to the manufacturer’s instructions. The entire volume of Cy3-labeled RNA was incubated at 100 °C for 5 min in a 45 μl reaction volume containing 1x Agilent blocking agent, Hybridization Spike-In and Hi-RPM Hybridization Buffer. Upon completion of this incubation, samples were hybridized to an 8×15 k customized Agilent Rat miRNA microarray containing both miRBase 15.0 and 16.0 probes for 20 h at 55 °C on a rotating rack in an Agilent hybridization oven. After hybridization, microarrays were washed for 5 min at room temperature with GE Wash Buffer 1 (Agilent Technologies) and then for 5 min at 37 °C with GE Wash buffer 2 (Agilent Technologies); slides were then dried immediately. Immediately after this washing and drying step, slides were scanned using an Agilent DNA Microarray Scanner (G2565AA) at the following settings: one-color scan for 8×16 k array slides, scan area, 61×21.6 mm; scan resolution, 5 μm; dye channel, green; and extended dynamic range scan mode (Hi=100%, Lo=5%).

Microarray data analysis

Expressionist analysis software Ver. 7.6 (Genedata AG, Basel, Switzerland) was used for normalization, principal component analysis (PCA) and hierarchical clustering. First, all signal intensities were scaled to the 75th percentile of the median of the dynamic target value for each array using the central tendency normalization method. Next, the following two statistical parameters were calculated for each probe: (i) P-value of the Shapiro-Wilk test with Bonferroni correction (R software) and (ii) maximal signal intensity. Probes were then filtered by a P-value of <0.05 and a maximal signal intensity of >100. To identify organ-specific miRNAs, expression profiles of filtered probes were further analyzed by model-based clustering using the ‘mclust’ R package (http://cran.r-project.org/web/packages/mclust/index.html). For this analysis, a histogram was created to determine the distribution of expression values among different organs for each gene. For multiple distributions, the presence of an isolated peak was considered attributable to organ-specific expression of miRNAs. Genes with isolated peaks consisting of less than 37 microarrays (20% of all microarrays) in the histogram were selected as candidate miRNAs that were specifically expressed in one or a few organs.

miRNA microarray experiments

In this study, microarray experiments were performed according to the manufacturer’s instructions, and the quality of each experiment was assessed in QC reports generated from the Agilent microarray systems.

Possible effects of peripheral blood cell-derived miRNAs

To evaluate the possible effects of peripheral blood cell-derived miRNAs on the miRNA expression profiles of organs, two different sampling conditions, with or without perfusion, were compared to assess effects on miRNA expression profiles in four organs (heart, kidney, liver and lung). As shown in Figure 1, blood-cell–specific miRNAs (e.g., miR-451 and miR-144)²¹ were clearly evident in nonperfused samples, but not in perfused samples. On the other hand, for each organ, miRNA profiles for perfused samples were highly correlated with those for nonperfused samples (R²>0.995). These data support the hypothesis that there are no distinct differences in miRNA profiles between these major organs and peripheral blood cells with some exceptions, such as miR-451 and miR-144, and that expression profiles for the majority of miRNAs were not markedly affected by remaining peripheral blood. Moreover, our data showed that miR-451 and miR-144 were specifically expressed in hematopoietic tissues e.g., spleen and bone marrow (refer to the original dataset for details). Therefore, microarray data obtained from nonperfused samples were analyzed in combination with those from perfused samples when generating the final comprehensive dataset.

Principal component analysis (PCA)

PCA was performed to comprehensively compare miRNA expression profiles among all organs in the entire experimental dataset (Figure 2a). Along the principal component (PC) 1 axis, which accounts for 29.1% of all variation, the clusters of signals from nervous system (brain, spinal cord, optic nerve and sciatic nerve; shown in blue colors), pituitary gland and adrenal gland samples were clearly separated from intestine samples (shown in red), whereas along the PC2 axis (11.4%), muscle (shown in green) samples were clearly separated from nervous system and intestine samples. Along the PC3 axis (8.1%), the clusters of signals from lymph and hematopoietic tissue (shown in pink) samples were clearly separated from those of other samples. Similarly, along the PC4 axis (6.7%) and PC5 axis (5.0%), the clusters of signals from pancreas and lung samples, and those from reproductive organ samples, were clearly separated from others. A tendency towards densely clustered signals from triplicate samples of the same organ was observed in PCA results. Additional information and eigenvalues of PC1 to PC5 axes are summarized in Tables 2 and 3 (available online only).

Furthermore, miRNA microarray data obtained from digestive organs, the nervous system and skeletal/smooth muscles were extracted from the whole dataset and subjected to system-specific PCA (Figure 2b). In the intestinal-specific subanalysis, clusters of signals from upper digestive organ (esophagus and stomach) samples were clearly separated from those of lower digestive organs (small and large intestine) along PC1 (37.2%), whereas along the PC2 axis (10.8%), those from small and large intestine samples were further divided into each organ by functional anatomy of the digestive system. In the nervous system subanalysis, a clear separation of clusters of signals were observed along both PC1 (35.7%) and PC2 (13.2%); those from cerebrum samples were clearly separated from ischial nerve samples along PC1, and those from the spinal cord were separated from optic nerve samples along PC2 (13.2%). Skeletal/smooth muscles were separated into four clusters: skeletal muscles, smooth muscles (blood vessel), smooth muscles (bladder and stomach) and others (esophagus, tongue and skin) (31.0% for PC1 and 16.4% for PC2). Overall, PCA successfully showed the similarity in gene expression profiles among functionally similar organs. These results provided evidence that the microarray data were of high enough quality to assess similarities and differences among miRNA profiles from different organs and tissues. In addition, the global miRNA expression profiles for these 55 normal organs included high-quality, reliable data that reflected the biological function of each organ.

Organ specific miRNA selection

To identify organ-specific miRNAs, all probes with any detectable signal were further filtered by a statistical analysis with criteria described in the Methods section; 296 probes met these criteria and were further analyzed using model-based clustering. In the model-based clustering, 128 probes were identified as miRNAs that were specifically expressed in one or more organs; further details on the statistical parameters calculated are available in Table 4 (available online only). Figure 3 shows a 2-dimensional hierarchical clustering diagram of groups of organs; the clustering was based on the expression profiles of the 128 filtered probes. Triplicate samples of the same organ were densely clustered; moreover, functionally similar organs were closely clustered to each other. Among these clusters, several miRNAs that were specifically expressed in specific organ(s) were successfully identified. Expression profiles of several representative organ-specific miRNAs are summarized (Figure 4 and Table 5 (available online only)). As shown in Table 5, more than half of these miRNAs were expressed in the corresponding human organs/tissues^{22–31,22–31,22–31,22–31,22–31,22–31,22–31,22–31,22–31,22–31}.