miRNAs in Blood Microvesicles Figure 2. Distribution of miRNA expression. The box plots were generated to demonstrate the distribution of miRNA expression from each PBMC and plasma microvesicle sample. The bottom and top of the boxes are the 25th and 75th percentile (the lower and upper quartiles, respectively), and the band near the middle of the boxes are the 50th percentile (the median). The ends of the whiskers represent the minimum and maximum of the data. (A) Raw CT score of the data from individual donor were plotted. (B). 18s standardized data (DCT ) were plotted: DCT = raw CT of PBMC or plasma microvesicles – CT of 18S. (C). Median normalized data of each donor was used. doi:10.1371/journal.pone.0003694.g002 between plasma microvesicles and PBMC based on their relative expression to the overall miRNA expression on each array using median normalization analysis (Figure 2C) [33]. The filtered and normalized data were subjected to hierarchical cluster analysis comparing the miRNA expression profile between the PBMC and plasma microvesicles samples (Figure 3). All but three PBMC samples clustered separately from the microvesicle samples indicating that the miRNA expression profile between the two groups was significantly different. It is not readily apparent why three samples clustered differently. These donors varied in age and gender. However, based on filtering criteria, we found 104 and 75 miRNAs were significantly expressed in microvesicle and PBMC samples, respectively (Figures 4A and 4B). Of these miRNAs, 71 were co-expressed among each sample group (Figure 4C). Age and gender effects We did not observe age and/or gender effects in miRNA expression from either sample group or across PBMC and plasma microvesicles samples. Notably, the median age for both female and male donors was 29 years. The oldest individual was 58 years old, while the youngest was 21 years of age. Thus, we further stratified the data to examine differences. For each PBMC and microvesicles compartment, we also compared the miRNA expression of the donors with the upper quartile of age to those with the lower quartile of age (mean age for each group was 48.966.2 and 21.961.2, respectively). However, we failed to detect significant differences in miRNA expression between the samples sets based on age (data not shown). Comparison of miRNA expression in PBMC and microvesicles In Table 1, miRNA expression was analyzed separately in plasma microvesicles and PBMC to determine the most highly expressed miRNAs and to determine the frequency of expression across all donors. Normalized expression for all miRNAs in the plasma microvesicles and PBMC from all tested individuals can be found in Table S1. miR-223, -484, -191, -146a, -16, -26a, -222, -24, -126, and -32 were detected in plasma microvesicles, while miR-150, -146b, PLoS ONE | www.plosone.org 4 November 2008 | Volume 3 | Issue 11 | e3694