Transcription profiling by array of a single human islet extracellular vesicle sample to study diabetes genes
ABSTRACT: Identification of transcriptional profile of several genes involved in diabetes in islet-derived extracellular vesicles (Evs). Recently, EVs are identified as a new mechanism in cell-to-cell communication by transfer of protein and genic information (mRNA, microRNA). Their role is under investigation in immunology, stem cell and cancer, but not in islets and diabetes. The aim of this experiment is to identify mRNA transcripts (in particular, mRNA transcripts involved in diabetes pathophysiology) present in islet Evs.
Project description:Tubular endothelial cells were cultured in hypoxic conditions (1% O2) and treated or not with microvesicles derived from endothelial progenitor cells.<br>mRNA profiling of hypoxic cells, treated or not with MVs, was analyzed after nromalization with mRNA profiling of normoxic cells.<br><br>This experiment was updated on 27th Jan 2011 to correct descriptions.
Project description:Human pancreatic islets were isolated from pancreas of deceased donors by Ricordi's procedure and cultured in CMRL 1066 medium additioned with human albumin. EVs were isolated from conditioned medium derived from islet culture after isolation. Once isolated, RNA of islets and islet-derived EVs was extracted and analyzed for microRNA expression within 48 hours after isolation.
Project description:Glomerular endothelial cells were cultured in normal condition and treated or not with microvesicles derived from endothelial progenitor cells. mRNA profiling of glomerular endothelial cells , treated with MVs, was analyzed after normalization with mRNA profiling of untreated cells.
Project description:HUVECs were cultured in hypoxic conditions for 24 hours and treated or not with 1uM caffeic acid. Then cells were evaluated for expression of different genes involved in angiogenesis
Project description:In this study, we address mRNA composition of hepatocyte-like derived extracellular vesicles (EVs), using as cellular model the mouse liver derived cell line MLP29, and primary cell culture of rat hepatocyte (RH) obtained by in vivo liver perfusion. The study shows qualitative characterization of RNA, identification of transcripts and its functional characterization through gene expression array technique. To reach a deeper nowledge in the biology of EVs, we perform RNase protection assay, density gradients matching RNA with typical exosomal protein markers, and capture assays to probe that mRNA was internalized. Aim of the project: To identify transcripts present in extracellular vesicles secreted by Rat hepatocytes primary cell culture and to identify extracellular vesicles secreted by mouse hepatocyte cell line MLP29, and in this case, compare the enrichment of transcripts respect to the cell, to know if the composition in the extracellular vesicles is similar to the cell, or if their composition is not directly determined by the abundance of transcripts in the cell.
Project description:TLR3, TLR7, and TLR9 stimulation induces many mouse inflammatory and autoimmune cytokines or immune receptors DRGN were cultures 5 days prior to a 16 hour stimulation - Three separate studies were analyzed for inflammatory response
Project description:The current project is within the range of molecular immunogenetic auto immune diseases and refers to the comparative study of promiscuous gene expression of tissue-specific antigens (TSAs) in the thymus of NOD mice line (non obese diabetic) who plays the auto-immune diabetes mellitus type 1, during the transition from state pre-diabetics to diabetics. Use the technology of oligo arrays to investigate the expression of miRNAs and cDNA microarrays to investigate the expression of genes encoding the messenger RNAs including TSAs (tissue specific antigens). Use the technology of oligo arrays to investigate the expression of miRNAs and cDNA microarrays to investigate the expression of genes encoding the messenger RNAs including TSAs (tissue specific antigens). Female NOD mice were born in specific pathogen free (SPF) conditions at the CEMIB-UNICAMP animal facility (University of Campinas, SP, Brazil) and maintained in SPF mini-isolators in our laboratory at the University of Sï¿½o Paulo, Campus of Ribeirï¿½o Preto, Brazil. We studied both pre-diabetic (8ï¿½2 week-old) and diabetic (20ï¿½2 week-old) animals. Diabetes was confirmed by blood glucose levels (?250 mg glucose/dL) using the Accu Check ï¿½ Active Kit (Roche Diagnï¿½stica Brasil, Sï¿½o Paulo, Brazil). The thymic stroma was separated from the whole thymus, as previously described (Gray et al. 2002). The central idea is to trace signatures of differential gene expression of the thymus at different stages (transition from state pre-diabetics to diabetics) and, using bioinformatics programs, applied to the analysis of data from arrays [Cluster & Tree View (for signatures of expression), SAM (for statistical analysis of the miRNAs and differentially expressed genes from TSAs), GenMiR++ and Cytoscape (to establish networks between miRNAs genes and genes of TSAs)].