Extracellular Vesicles in Luminal Fluid of the Ovine Uterus
ABSTRACT: 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.
Project description:RNA seq analysis was conducted to determine gene expression in the day 14 ovine conceptus. This was used in conjunction with the day 14 PPARG ChIP-seq analysis to identify genes bound by PPARG which were also expressed or not expressed in the day 14 conceptus. Understanding changes in gene expression during early pregnancy is critical to improving fertility and reproductive efficiency in ruminants. RNA seq analysis of 4 conceptuses from 4 individual Day 14 pregnant columbia/rambouillet crossbred ewes
Project description:MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486. The hearts of 3 male 8 month old Sprague-Dawley rats were rapidly extracted after euthanasia with sodium pentobarbital. A section of the free wall of the left ventricle was dissected into epicardium, mid-myocardium and endocardium by cutting approximately 1 mm from the epicardial and endocardial surfaces. Small RNA was extracted (miRNeasy Kit; Qiagen, Crawley UK), quantified (Nanodrop; Thermo Scientific) and quality assessed for degradation (RNA Nano Chip, Bioanalyser 2100; Aligent Technologies, Wokingham UK; only samples with a RNA integrity no. (RIN) ≥8 were carried forward) and retention of small RNA (Small RNA Chip, Bioanalyser 2100). Small RNA was preferentially ligated with adapters, reverse transcribed into cDNA and amplified with 9 individually tagged primer indices (TruSeq Small RNA Sample Preparation Kit; Illumina, Little Chesterford, UK) and a library of small RNA created for each sample. After gel purification the cDNA products were again analysed on the bioanalyser using a High Sensitivity DNA Chip and assessed for the presence and concentration of the peak corresponding to ligated and tagged miRNA (approximately 147nt). Only samples with suitable RIN values exhibiting good retention of small RNA species were used for library preparation. After pooling, the samples were sequenced by TrinSeq (Trinity Genome Sequencing Lab & Neuropsychiatric Genetics Group, Trinity College Dublin, Ireland (http://www.medicine.tcd.ie/sequencing); using TruSeq SR Cluster Kit v5 (Illumina) and the resultant data trimmed and aligned to miRBase v18 (CLC Genomics Workbench v4.0; CLC bio, Swansea UK).
Project description:MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs. The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Cells were transfected with a plasmid to direct overexpression of miR-146a. Extracellular vesicles were isolated by ultracentrifugation from untreated and transfected cells. RNA was isolated from one sample each of untreated and transfected cells and vesicles.Small RNA libraries were prepared for sequencing.
Project description:We report the application of small RNA sequencing technology for high-throughput profiling of microRNA molecules (miRNAs) in blood leukocytes of Parkinson's Disease (PD) patients. Expression changes which may contribute to PD pathogenesis and reaction to treatment were identified in RNA preparations from blood leukocyte samples collected from PD patients prior to and following DBS neurosurgery on stimulation and following a short one hour cessation of the electrical stimulation (which enabled dissociation of the surgery effects from the electrical stimulation alone) and from matched healthy control volunteers, all under signed informed consents. Examination of four states: Parkinson's Disease (PD) patients 1 day prior to DBS, PD patients a few weeks after DBS neurosurgery (upon symptoms stabilization) both on and following one hour electrical stimulation cessation, and age-matched healthy control volunteers.
Project description:ChIP-seq analysis of HepG2 cells revealed that many of the target genes of LSD2 were related to lipid metabolism. We found that LSD2 is an important epigenetic regulator of hepatic lipid metabolism. Examination of LSD2/DNA interaction in HepG2 cells in normal condition.
Project description:Staphylococcus aureus is an important human pathogen that causes life-threatening infections, and is resistant to the majority of our antibiotic arsenal. This resistance is complicated by the observation that most antibacterial agents target actively growing cells, thus, proving ineffective against slow growing populations, such as cells within a biofilm or in stationary phase. Recently, our group generated updated genome annotation files for S. aureus that not only include protein-coding genes but also regulatory and small RNAs. As such, these annotation files were used to perform a transcriptomic analysis in order to understand the metabolic and physiological changes that occur during transition from active growth to stationary phase; with a focus on sRNAs. We observed ∼24% of protein-coding and 34% of sRNA genes displaying changes in expression by ≥3-fold. Collectively, this study adds to our understanding of S. aureus adaptation to nutrient-limiting conditions, and sheds new light onto the contribution of sRNAs to this process. Bacterial cells were grown in TSB medium at 37°C with shaking for 3h (exponential growth phase) or 16h (stationary growth phase).
Project description:We aimed to determine the binding sites of the putative transcriptional regulator PafBC under DNA damage stress induced by mitomycin C or under oxidative stress induced by hydrogen peroxide. Therefore, we chose a ChIP-seq approach, crosslinking cells before PafBC-DNA complexes were immunoprecipitated with a PafBC-specific antibody.
Project description:ChIP-seq analysis of LSD2-depleted HepG2 cells revealed that many of the target genes were related to lipid metabolism. We found that LSD2 is an important epigenetic regulator of hepatic lipid metabolism. Examination of LSD2/H3K4me1 interaction in control and LSD2-knockdowned HepG2 cells.
Project description:In this study, we have investigated the physiological consequences of PHB (poly(3-hydroxybutyrate)) synthesis in H. seropedicae by characterising the trancriptional changes in a mutant strain lacking phaC1 gene which codes for the PHA synthase enzyme essential for the last step in the synthesis of PHB. To do this experiment both wild type (SmR1) and phaC1 mutant were cultured on Nfb-Malate-HP media suplemented with 20 mM of ammonium chloride under 30 Celsius degrees and 120 rpm shaking rate until the late log-phase (O.D600 = 1.0), when the peak of PHB production is observed. The cultures obtained as above were harvested for RNA extraction and transcriptome analysis.
Project description:In recent years, the Gram-negative bacterium Acinetobacter baumannii has garnered considerable attention for its unprecedented capacity to rapidly develop resistance to antibacterial therapeutics. This is coupled with the seemingly epidemic emergence of new hyper-virulent strains. Although strain-specific differences for A. baumannii isolates have been well described, these studies have primarily focused on proteinaceous factors. At present, only limited publications have investigated the presence and role of small regulatory RNA (sRNA) transcripts. Herein, we perform such an analysis, describing the RNA-seq-based identification of 78 A. baumannii sRNAs in the AB5075 background. Together with six previously identified elements, we include each of these in a new genome annotation file, which will serve as a tool to investigate regulatory events in this organism. Our work reveals that the sRNAs display high expression, accounting for >50 % of the 20 most strongly expressed genes. Through conservation analysis we identified six classes of similar sRNAs, with one found to be particularly abundant and homologous to regulatory, C4 antisense RNAs found in bacteriophages. These elements appear to be processed from larger transcripts in an analogous manner to the phage C4 molecule and are putatively controlled by two further sRNAs that are strongly antisense to them. Collectively, this study offers a detailed view of the sRNA content of A. baumannii, exposing sequence and structural conservation amongst these elements, and provides novel insight into the potential evolution, and role, of these understudied regulatory molecules. This study is based on the annotation of novel sRNAs on basis of an Acinetobacter baumannii RNA sequencing dataset. Each sample was generated by pooling three independent biological replicate RNA preps