Plasma Microvesicles miRNA miR-223 miR-484 miR-191 miR-146a miR-016 miR-026a miR-222 miR-024 miR-126 miR-032 Normalized Expression (6S.D.) 15896653 50.9622.9 46.4614.9 39.5619 25.4613.3 25.269.95 24.5612.4 22.7610.5 18.268.04 15.3632.6 Frequency Detected Among Donors (%) Top Ten Ranking (%) 100 100 100 100 100 100 100 100 100 100 100 96 100 88 78 90 76 80 66 31 PBMC miRNA miR-223 miR-150 miR-146b miR-016 miR-484 miR-146a miR-191 miR-026a miR-019b miR-020a Normalized Expression (6S.D.) 21436499 241694.6 57.5621.1 54.7632.9 40.6618.8 39.6613.0 32.4615.6 3068.92 21.767.49 1565.11 100 98 100 100 89 100 100 100 100 100 Table 1. Normalized expression and rank analysis of miRNAs in plasma microvesicles or PBMC. Expression of miRNAs in the plasma microvesicles and PBMC were analyzed separately (n = 51). The normalized miRNA expression was generated using median normalization analysis then the mean CT value was converted to expression (2^(2DCT)) 6standard deviation (S.D.). Shown is the ‘‘Frequency Detected Among Donors’’ represents the percentage of donors in which the miRNA was detectable by qRT-PCR. The ‘‘Top Ten Ranking’’ indicates the percentage of individuals in which the normalized expression for each miRNA fell within the top ten highly expressed miRNAs. doi:10.1371/journal.pone.0003694.t001 the highest expressed miRNA in platelets. Of interest, we failed to observe the expression of miR-484 in the platelets, which is found in both plasma microvesicles and PBMC. Discussion In this study, we hypothesized that miRNAs circulate in plasma microvesicles, platelets, and PBMC of normal human volunteers in the peripheral blood. We have characterized peripheral blood miRNA patterns in healthy humans. We found significant differences in miRNA expression between plasma microvesicles, platelets, and PBMC. To date, numerous studies have established the ability for miRNAs to regulate many cellular functions. However, these studies largely imply that the miRNA resides within its host cell to elicit an effect [39]. Our data indicate that the miRNAs are also contained in plasma microvesicles that may influence homeostasis. The defined mechanism or cellular signals that regulate microvesicle production is unknown. Some suggest that the vesicle ‘‘hijacks’’ the cytoplasm prior to release from the cell through membrane blebbing [23]. This paradigm suggests that packaging of the miRNAs in the microvesicle may be random. Alternatively, endosomal trafficking may modulate the formation of microves- icles [40]. Since P-bodies in the cytoplasm co-localized with miRNAs [2,4], we are exploring whether P-bodies may regulate the transfer and sorting of miRNAs into vesicles. There are other reports of tumor cell fragments containing miRNAs in the plasma and/or serum [41,42]. Recently Taylor and colleagues found circulating tumor microvesicles in the peripheral blood of patients with ovarian cancer but not healthy individuals [41]. These microvesicles express miRNAs found in the primary tumor. It is conceivable that the miRNAs in the plasma microvesicles circulate to tissue targets. Further examination of the highest expressed miRNAs in plasma microvesicles (Table 1), demonstrate that many of these miRNA are predicted to regulate hematopoi- esis and cellular differentiation. For instance, expression of miR- 223 is predicted to influence myeloid, granulocyte and osteoclast differentiation [43–45]. miR-223 also appears to have a role in hematopoietic stem cell proliferation [43]. Interestingly, miR-223 expression is lost in acute myelogenous leukemia (AML) [46]. miR-24 expression is regulated by TGF-b, a potent positive and negative regulator of hematopoiesis [47,48]. Both miR-24 and miR-16 regulate red cell production [49,50], while miR-16 also modulates lymphoid development [51]. Loss of miR-16 expression has been extensively examined in chronic lymphocytic leukemia (CLL) [52,53]. In contrast, downregulation of miR-126 expression occurs during megakarocyte differentiation [54]. miRNAs in Blood Microvesicles Frequency Detected Among Donors (%) Top Ten Ranking (%) 100 98 100 100 88 98 94 100 80 4 p-value 1.47E-02 2.07E-02 2.23E-02 2.45E-02 2.78E-02 Table 2. Predicted pathways regulated by miRNAs expressed in the plasma microvesicles and PBMC fractions. Plasma Microvesicles glycerophospholipid metabolism inositol phosphate metabolism phospholipid degradation alanine and aspartate metabolism estrogen receptor signaling p-value 3.29E-03 5.77E-03 9.17E-03 1.96E-02 2.14E-02 PBMC axonal guidance signaling synaptic long-term potentiation estrogen receptor signaling glycerophospholipid metabolism D-glutamine and D-glutamate metabolism Predicted targets were compiled separately using Sanger miRBase and TargetScan for the top nine miRNAs expressed in the plasma microvesicles and PBMC (Table 1). The common predicted targets were subsequently analyzed by IPA software to determine predicted pathways regulated by the miRNAs. Fisher’s exact test was used to calculate a p-value determining the probability of the association between the genes in the dataset and the canonical pathway. doi:10.1371/journal.pone.0003694.t002 PLoS ONE | www.plosone.org 7 November 2008 | Volume 3 | Issue 11 | e3694