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Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells. To characterize the RNA-content of the blood plasma sub fractions, we performed high throughput sequencing of the RNA pelleted within vesicles at 16,000 g and 160,000 g as well as RNA remaining in the vesicle-free supernatant. We found that blood plasma sub fractions contain not only extensive set of microRNAs but also fragments of other cellular RNAs: rRNAs, tRNAs, mRNAs, lncRNAs, small RNAs including RNAs encoded by mtDNAs. Our data indicate that a variety of blood plasma RNAs circulating within vesicles as well as of extra-vesicular RNAs are comparable to the variety of cellular RNA species.

Understanding structures of circulating RNA expands fundamental knowledge of cell communications and signaling pathways as well as allows developing new molecular diagnostic approaches. The aim of this study was to deploy a new approach to sequencing cDNA library construction which expands the capabilities of high-throughput sequencing analysis of small non-coding RNAs. With the approach, we performed massively parallel sequencing of human blood plasma RNA to document profile of common and peculiar RNA species normally circulating in blood of healthy individuals.Total RNA was extracted from blood plasma samples of eight apparently healthy individuals. To obtain comprehensive cDNA libraries RNA was dephosphorylated and then 5'-phosphorylated. 5'-Phosphorylated total plasma RNA was ligated with adapters, reverse transcribed and eight personalized cDNA libraries were constructed. Libraries were sequenced with SOLiD(™) technology.Fragments of rRNA, mitochondrial transcripts, microRNAs, fragments of scRNAs, snRNA and snoRNA, fragments of several mRNAs as well as the set of newly discovered transcripts were found to be permanent representatives of human blood plasma RNAs. Advanced mapping allowed to identify circulating herpes virus and enterobacterial transcripts. Documented profile of circulating RNA of healthy individuals provides basis for development of new approaches in research and diagnosis of human pathology.

Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells. To characterize the RNA-content of the blood plasma sub fractions, we performed high throughput sequencing of the RNA pelleted within vesicles at 16,000 g and 160,000 g as well as RNA remaining in the vesicle-free supernatant. We found that blood plasma sub fractions contain not only extensive set of microRNAs but also fragments of other cellular RNAs: rRNAs, tRNAs, mRNAs, lncRNAs, small RNAs including RNAs encoded by mtDNAs. Our data indicate that a variety of blood plasma RNAs circulating within vesicles as well as of extra-vesicular RNAs are comparable to the variety of cellular RNA species.

We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 µm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 µm due to used laser wavelength of 0.66 µm.

Promoter methylation rates of three tumor suppressor genes were detected in the total cirDNA, derived from plasma and cell-surface-bound fractions, of gastric cancer patients (n = 30) and healthy subjects (n = 30) using MS-PCR. The tumor marker protein levels (CEA, CA 72.4, CA 19.9) were assessed in plasma samples by commercial immunoassay kits. Methylated forms of p15, MGMT and hMLH1genes were detected in the total cirDNA with high rates at II, III and IV stages of gastric cancer. Sensitivity of the MS-PCR-based assay for at least one of methylated p15 and hMLH1 genes in gastric cancer was found to be much higher compared with the sensitivity of the immunoassay for elevated levels of CA 72.4 and CA 19.9 proteins (63 and 30%, respectively). No significant correlation was found between epigenetic and protein markers so indicating their independent development in gastric tumor pathogenesis. To conclude, epigenetic alterations of total cirDNA and elevated levels of tumor-associated proteins were found to be independently associated with gastric cancer indicating their usefulness as complementary diagnostic and prognostic markers for gastric cancer.

The concentration of circulating DNA (cirDNA) in blood plasma and cell-surface-bound fractions of lung cancer patients and healthy individuals was measured using real-time PCR for the single-copy β-actin gene and LINE-1 repetitive elements. The average concentration of cirDNA in plasma was shown to be similar in healthy individuals and non-small cell lung cancer (NSCLC) patients. However, the concentration of cell-surface-bound circulating DNA (csb-cirDNA) in NSCLC patients was significantly lower than that found in healthy individuals (P = 0.009 and P = 0.002 for β-actin and LINE-1 assays, respectively). The decrease of csb-cirDNA concentration in NSCLC patients was associated with a poor disease prognosis. The ratio of the β-actin gene to LINE-1 fragments in the csb-cirDNA was found to be elevated in NSCLC patients compared with control (3.4 and 1.7 respectively, P = 0.007). Thus, in lung cancer patients the cirDNA quantification by PCR for β-actin gene and LINE-1 fragments was found to provide a subsidiary data for tumor detection and prognosis.

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.