Recent evidence reveals that genetic exchange of mRNA and miRNA between cells can be accomplished through microvesicles, or exosome-mediated transfer [20]. Microvesicles are small exosomes/vesicles of endocytic origin released by normal healthy or damaged cell types [21–23]. Alternative names for microvesicles provide insight into their source. Microparticles are microvesicles released from activated platelets; exosomes are microvesicles secreted by tumors; and ectosomes are neutrophil-derived microvesicles [23]. Current estimates of microvesicle concentra- tion in the peripheral blood of healthy individuals are 5–50 mg/ml. Microvesicles are shed from the plasma membrane into the extracellular environment to facilitate communication between cells. Despite their small size (50 nm to 1 mm) microvesicles are enriched in bioactive molecules and contain nucleic acid and/or protein; these cell particles play a role in growth, differentiation and cancer progression [24]. Tumor-derived microvesicles transfer mRNA to monocytes within the tumor microenvironment to activate these cells to produce cytokines to enhance tumor growth and dampen the immune response [25]. Deregibus et al report that mRNA from endothelial-derived MV are pro-angiogenic [26]. In the peripheral blood, two-thirds of microvesicles are derived from platelets. Platelet-derived microvesicles play a role in angiogenesis and the metastatic spread of cancers such as lung cancer [27]. Platelet-derived microvesicles induce an immune response upon regulating gene expression in hematopoietic, endothelial, and monocytic cells [28,29]. Notably, platelet-derived microvesicle subpopulations are increased in patients with sepsis [30,31], whereas patients with pulmonary arterial hypertension have increased endothelial-derived microvesicles [32]. However it is currently unknown whether microvesicle content changes in theses diseases. Interestingly, a connection between microvesicles and miRNA has been recently made. Recently, Valadi and colleagues reported that vesicles released from human and murine mast cell lines contain over 1200 mRNA and approximately 121 miRNA molecules [20]. Considerable evidence demonstrates the importance of miRNA as an inevitable cornerstone of the human genetic system. Employing the use of microvesicles to transfer genetic material would be an efficient transfer method within the human body. Microvesicles containing miRNAs would enable intercellular and inter-organ communication in the body. Here, we explored the hypothesis that miRNAs are contained within microvesicles in the peripheral blood. We examined the expression of miRNAs from the peripheral blood microvesicles and PBMC from normal human volunteers. Our study identifies miRNAs in circulating microvesicles in the plasma and in PBMC and characterizes specific miRNA expression. Methods Blood collection and microvesicle isolation Peripheral blood (40 cc) was collected in EDTA tubes from 24 female and 27 male healthy non-smoking Caucasian donors following informed consent. Written consent was obtained from all subjects in accordance with approved Institution Review Board protocol (1978H0059) and HIPAA regulations. Criteria for volunteers consist of no recent illness or treatment for a chronic medical condition. No medical history was obtained from donors. Collection of the blood occurred between morning and early afternoon. The average age for female and male donors was 35612 and 3169, respectively, while the median age for both female and male donors was 29. The peripheral blood was diluted 1:1 with sterile low endotoxin PBS (Mediatech, Inc. Manassas, miRNAs in Blood Microvesicles VA), layered over ficoll-hypaque (d = 1.077) (Mediatech, Inc.), and centrifuged as previously described [33]. The mononuclear cell fraction was washed once in PBS. The microvesicles were purified from the plasma. Briefly, the vesicles were concentrated by centrifugation at 160,0006 g for 1 hr at 4uC [34]. To isolate platelets, blood was collected in 3.8% sodium citrate tubes from six donors, using a washing method as previously described [35,36]. Following centrifugation of the blood, the platelet rich plasma was incubated with 1 mM of PGE1 (Sigma-Aldrich, St. Louis, MO). The platelets were washed twice in Tyrodes buffer containing 138 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, 0.36 mM NaHPO4, 5.5 mM glucose, 1.8 mM CaCl2, and 0.49 mM MgCl2, pH 6.5. The platelets were then washed one additional time in Tyrodes buffer, pH 7.4. RNA Extraction Total RNA was isolated by Trizol (Invitrogen, Carlsbad, CA) extraction method. To increase the yield of small RNAs, the RNA was precipitated overnight. RNA concentration and RNA integrity were determined by capillary electrophoresis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc, Santa Clara, CA). We recovered an average 1.740 mg of total RNA from the plasma fractions. For RNA isolated from mononuclear cells, only a RNA integrity number (RIN)$9 was used along with its matched plasma sample for profiling. Since the intact 18s and 28s rRNA were variable in the microvesicles, the RIN was not a constraint for these samples, however we observed a RIN between 2–8. miRNA profiling by quantitative PCR At the time of the study, commercially available primers were available for 420 mature human miRNAs. We used these looped primers to profile 420 mature miRNAs by real-time PCR using an Applied Biosystems 7900HT real-time PCR instrument equipped with a 384 well reaction plate. RNA (500 ng) was converted to cDNA by priming with a mixture of looped primers (Mega Plex kit, Applied Biosystems, Foster City, CA) using previously published reverse transcription conditions [37]. As there is no known control miRNA in microvesicles, several internal controls were examined. Primers to the internal controls, small nucleolar (sno)RNA U38B, snoRNA U43, small nuclear (sn)RNA U6 as well as 18S and 5S rRNA were included in the mix of primers. Liquid- handling robots and the Zymak Twister robot were used to increase throughput and reduce error. Real-time PCR was performed using standard conditions. Flow Cytometry Peripheral blood microvesicles were directly immunostained from plasma without concentration by centrifugation. To determine the cellular origin, 0.5 cc plasma was immunostained per panel of antibodies. Panel I contained antibodies recognizing CD66b-FITC (neutrophil) (BD Biosciences, San Jose, CA), CD202b (Tie2)-PE (endothelial) (R&D Systems, Minneapolis, MN), CD206 PE-Cy5 (macrophage/dendritic) (BD Biosciences), CD79a-APC (B-cell) (BD Biosciences), and CD14 Pe-Cy7 (monocyte) (BD Biosciences). Panel II contained antibodies to CD41a-PE-Cy5 (platelet) (BD Biosciences), CCR2-APC (mono- cyte) (R&D Systems), CCR3-PE (dendritic cell) (Caltag, Carlsbad, CA), CCR5-PE-Cy7 (macrophage) (BD Biosciences), and CD3- Alexa 610 (T-cell) (BD Biosciences). Panel III contained isotype control antibodies (BD Biosciences). All antibodies were used according to manufacturers’ recommendation. To confirm that the microvesicles were the correct size, flow cytometry gates were set using 2 micron bead standards (BD Biosciences). The samples PLoS ONE | www.plosone.org 2 November 2008 | Volume 3 | Issue 11 | e3694