RESUMEN
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.
Asunto(s)
Vesículas Citoplasmáticas/genética , Vesículas Extracelulares/genética , ARN/genética , ARN/aislamiento & purificación , Centrifugación/métodos , Vesículas Citoplasmáticas/metabolismo , Vesículas Extracelulares/metabolismo , Citometría de Flujo , Humanos , Integrina alfa2/sangre , MicroARNs/sangre , MicroARNs/genética , MicroARNs/aislamiento & purificación , ARN/sangre , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , ARN Largo no Codificante/aislamiento & purificación , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Ribosómico/sangre , ARN Ribosómico/genética , ARN Ribosómico/aislamiento & purificación , ARN de Transferencia/sangre , ARN de Transferencia/genética , ARN de Transferencia/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Plaquetas/química , Plaquetas/citología , Citometría de Flujo/métodos , Simulación por Computador , Bases de Datos Factuales , Humanos , Luz , Dispersión de RadiaciónRESUMEN
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.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patología , Citometría de Flujo/métodos , Fotometría/métodos , Refractometría/métodos , Humanos , Luz , Dispersión de RadiaciónRESUMEN
OBJECTIVE: 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. METHODS: 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. RESULTS/CONCLUSION: 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.