Exosomes mediate the cell-to-cell transmission of interferon alpha-induced antiviral activity_miRNA microarray
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ABSTRACT: LSECs were grown in culture medium supplemented with 10% FBS (depleted of endogenous exosomes by overnight centrifugation at 100,000 g) and were treated with or without 1,000 U/ml IFN-a (PBL Interferon Source, Piscataway, NJ, USA) for 48 h. The exosomes from the cell culture supernatants were isolated by differential centrifugation as follow, at 300g for 10 min, 2,000g for 10 min, 10,000g for 30 min, and 100,000g for 70 min, washed once with PBS, and purified by centrifugation at 100,000g for 70 min. Total RNA of purified exosomes were extracted and Exiqon miRCURY LNA Expression Array were used for detection of mRNA and miRNA, respectively.
Project description:Human liver sinusoidal endothelial cells (LSECs) were grown in culture medium supplemented with 10% FBS (depleted of endogenous exosomes by overnight centrifugation at 100,000 g) and were treated with or without 1,000 U/ml IFN- alpha (PBL Interferon Source, Piscataway, NJ, USA) for 48 h. The exosomes from the cell culture supernatants were isolated by differential centrifugation as follow, at 300g for 10 min, 2,000g for 10 min, 10,000g for 30 min, and 100,000g for 70 min, washed once with PBS, and purified by centrifugation at 100,000g for 70 min. Total RNA of purified exosomes were extracted and Agilent Human 4x44K Gene Expression Array.
Project description:Hybrid (Bound) and non-hybrid (Unbound) DNA from Hela EV was isolated using DRIP procedure as follows. Between 1-5μg of nucleic acid DNA from EVs (DNase0 treated) was incubated overnight with 1μg of anti-DNA-RNA Hybrid (S9.6) antibody (KeraFast) under rotation at 4°C in 500μl of binding buffer (10mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The following day, 25μl of A/G magnetic beads (Pierce™) were washed twice in 500μl of binding buffer (30' min each wash) and added for 2h at room temperature under rotation to the S9.6+nucleic acid complex. The nucleic acids bound to the S9.6 antibody (Hybrid fraction or Bound) were separated from those unbound (Non-Hybrid or Unbound) by using a magnetic rack (which captured the Bead+Hybrid fraction). 500μl of the unbound fraction (bead-free) were transferred into a new 1.5ml tube and collected for further analysis. The Bead+Hybrid fraction was washed twice in 250μl binding buffer for 15' min at room temperature under rotation. The hybrids were then eluted with 250μl of elution buffer (50mM TRIS pH 8.0, 10mM EDTA, 0.5% SDS) for 15' min at room temperature under rotation. The elution step was repeated twice to make sure that all the hybrid molecules were collected. As a control of all there experiments to make sure, we were isolating and precipitating Hybrid DNA:RNA molecules, samples were also pre-treated with RNaseH1 (RNaseH), which degrades the RNA filament only when present in the hybrid structure.
Project description:microRNA profiles of Exosomes from Pooled NPC Patients serum comparing Control Exosomes from Healthy donors serum Two-condition experiment, Exosomes from Pooled Healthy donors serum vs. Exosomes from Pooled NPC Patients serum. Biological replicates: 1 Exosomes from Pooled Healthy donors serum, 1 Exosomes from Pooled NPC Patients serum,
Project description:To determine the different gene signatures between primary tumor and tumor-derived exosomes, we have employed RNA-sequencing as a discovery platform to identify gene signatures of tumor-derived exosomes, taking the original tumors as a control. We subcutaneously inoculated C57BL/6 mice with Lewis lung carcinoma (LLC). Three weeks later, tumor tissues were cut and tumor-derived exosomes were isolated as described in the "treatment protocol". Then, both exosomal RNA and tumor RNA were extracted and sequenced. From sequencing, we found that exosomal RNAs showed quite different transcript profiles from tumor RNAs. Examination of different gene signatures between primary tumor and tumor-derived exosomes. 2 replicates each.
Project description:To study the pathogenicity of Salmonella Typhimurium (ST) in heterophils, a whole Salmonella genome array was used to analyze RNA isolated from Salmonella in vitro encountered with heterophils (30 min, 1 h, 2 h, and 3 h) or medium control (NS). Dual-color, direct comparisons were carried out between heterophils-encountered ST and non-encountered controls (30 min vs. NS (30M/NS), 1 h vs. NS (1HR/NS), 2 h vs. NS (2HR/NS) and 3 h vs. NS (3HR/NS)). Each comparison includes four biological replicates. Out of 4588 genes on an array, there were 605, 758, 944 and 945 genes differentially expressed in the comparisons of 30M/NS, 1HR/NS, 2HR/NS and 3HR/NS, respectively (P < 0.001, fold-change > 2). Most of the Salmonella genes associated with invasion function were found differentially expressed at 2HR/NS and 3HR/NS comparisons. These candidate genes include TypeIII-secreted protein effector sopE2, Invasion proteins (e.g. invA, invE, invG, invH), and Invasion genes tranction activator hilA. The results of continuous transcriptional changes provide important information to elucidate the pathogenicity of ST in heterophils. Dual-color, common reference comparisons were carried out between heterophils-encountered ST and non-encountered controls (30 min vs. NS (30M/NS), 1 h vs. NS (1HR/NS), 2 h vs. NS (2HR/NS) and 3 h vs. NS (3HR/NS)). Four biological replicates, with dye swap labeling, were included in each of comparisons. Background subtracted signal intensity were collected from 16 arrays and normalized before data analysis.
Project description:Francisella tularensis LVS was grown to mid log phase and culture split into two. One culture served as control and other added H2O2 to final concentration of 0.03%. After 20 min at 37 C, RNA was isolated from both cultures
Project description:We analysed differentiation of the EATRO1125 strain of Trypanosoma brucei brucei, which was first isolated in 1966 from a bushbuck (Tragelaphus scriptus) in Uganda (origin stated (Bouteillea, 1995) without an original reference.<br>To analyse gene expression, we isolated at least 3 x 10e8 trypanosomes at different differentiation states, using two independent biological replicates. Bloodstream forms were harvested at a density of 2 x 10e5/ml (low density, logarithmic growth), and 2 x 10e6/ml (high density, logarithmic growth). Cells were also taken immediately upon attaining the density of 2 x 106/ml, treated with 3 mM cis-aconitate and moved to a room at 27M-0C. Samples were taken 30 min, 60 min, 12h and 24h after this. At 24 h the cells were centrifuged, resuspended (at 27M-0C) in MEM-Pros medium, which contains proline as the major energy source. Samples were taken again at 48h and 72h. A culture that had been maintained for several weeks after transformation was used as a source of established procyclic trypanosomes.
Project description:Dendritic cells (DCs) are the most potent antigen (Ag)-presenting cells. Whereas immature DCs down-regulate T cell responses to induce/maintain immunological tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact and vesicle exchange. Transfer of nanovesicles (<100nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle microRNAs (miRNAs), and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, an hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, as they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and post-transcriptional regulation between DCs. The study has analyzed the microRNA content of 4 samples of immature exosomes, 4 samples of matures exosomes, 2 samples of immature bone-marrow-derived DCs, and 2 samples of mature bone marrow-derived DCs.
Project description:Transcript profiling analysis of fruits from a segregating PopDG population, which were grouped/selected according to extreme cold susceptibility and subjected to different times of cold storage