Project description:The dataset contains FASTQ files referring to the study "Small RNA sequencing from CSF extracellular vesicles - PD/CTR". For this project, RNA was isolated from CSF extracellular vesicles obtained by ultracentrifugation. Libraries were prepared with the TruSeq Small RNA library prep Illumina, and sequencing conducted in the Illumina HiSeq4000.
| EGAD00001006629 | EGA
Project description:extracellular vesicles small RNA sequencing
Project description:Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2-18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.
Project description:Extracellularly released particles, including membrane vesicles, have increasingly been recognized as important for bacterial community functions and host-interaction processes, but their compositions and functional roles differ between species and also between strains of the same species. In this study, we have determined the composition of membrane vesicles and protein particles identified in the cell-free pellets of two strains of Apilactobacillus kunkeei, a defensive symbiont of honeybees. The membrane vesicles were separated from the extracellular particles using density gradient ultracentrifugation. The peaks of the RNA and protein distributions were separated from each other and the highest concentration of RNA was observed in the fractions that contained the membrane vesicles while the highest protein concentration coincided with the fractions that contained extracellular particles. A comparative proteomics analysis by LC-MS/MS showed that 37 proteins with type-I signal peptides were consistently identified across the fractionated samples obtained from the cell-free pellets, of which 29 were orthologs detected in both strains. Functional predictions of the extracellular proteins revealed the presence of glycoside hydrolases, glycosyltransferases, giant proteins and peptidases. The extracellular transcriptomes mapped to a broad set of genes with a similar functional profile as the whole cell transcriptome. This study provides insights into the composition of membrane vesicles and extracellular proteins of a bee-associated symbiont.
Project description:Extracellular vesicles play a pivotal role in the intercellular communications influencing various physiological and pathological processes. They carry a range of biomolecular cargoes including small non-coding RNAs which could serve as potential diagnostic biomarkers and therapeutic targets. In the current study we applied Next Generation Sequencing to investigate small RNA profiles of erythrocytes (Rbc), platelets (Plt), leukocytes (CD45cells), two plasma fractions and blood cell-derived extra-cellular vesicles (EVs, such as CD41+, CD45+, CD235a+, CD146+) obtained using high-sensitivity fluorescence activated vesicle sorting from plasma of healthy donors. We analyzed the proportions of various small ncRNAs across samples and identified sample specific profiles of microRNA (miRNA) and transport RNA-derived fragments (tRFs). It was found that the cumulative small ncRNAs profiles of EVs originated from platelets, erythrocytes, and leukocytes, which are considered to be dominant among the vesicles circulating in blood, differ from small ncRNAs profiles of plasma fractions, which represent macrovesicles and exosomes in circulation. The proportion of miRNAs in sorted EVs was significantly lower compared to other samples while the proportion of tRFs was higher. Moreover, all sorted EVs carried mostly cell type non-specific miRNAs. Taking together, the results demonstrate that the combination of high-sensitivity fluorescence activated vesicle sorting with small-RNA sequencing technique is a powerful approach to analyze small ncRNAs profiles in cell specific vesicles.
Project description:Exosomes and microvesicles (i.e., extracellular vesicles; EVs) have been identified within ovarian follicular fluid, and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, it remains unknown if EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle. Examination of small RNA population in bovine follicular fluid extracellular vesicles isolated from antral follicles
Project description:Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. Methods: To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers.
Project description:Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated two distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analysed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the two exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.
Project description:Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades, and it represents one of the most commonly used reagents in life science research. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here we report that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, likely due to the confounding effect of the FBS. Furthermore, although the absolute amount of individual RNA species in FBS is low, they can be taken up by cultured cells and affect the results and interpretations of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA.
Project description:Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Extracellular vesicle RNA was pooled (n=4 per status) and analyzed for small RNAs by sequencing on the Ion Torrent PGM platform and analysis with CLC Genomics Workbench small RNA workflow based on the miRBase (Release 19) Bos taurus database. Small RNA analysis of day 14 uterine luminal fluid extracellular vesicles isolated from pregnant and cyclic ewes.