Metabolomics of mice inoculated with human microbiota
Ontology highlight
ABSTRACT:
ORGANISM(S): Mouse Mus Musculus
TISSUE(S): Blood
SUBMITTER: 
Maureen Kachman
PROVIDER: ST000745 | MetabolomicsWorkbench | Mon Jun 19 00:00:00 BST 2017
REPOSITORIES: MetabolomicsWorkbench
Similar Datasets
Project description:Analysis of expression quantitative trait loci (eQTLs) using RNA derived from freshly harvested peripheral blood CD4+ lymphocytes from 200 asthmatics collected in clinical settings. RNA was obtained from peripheral blood CD4+ lymphocytes at four study centers. Peripheral blood (17 cc) was collected into BD Vacutainer CPT tubes (BD Diagnostics, Franklin Lakes, New Jersey) and placed on ice. Samples were centrifuged within 1 hour of collection for 20 minutes at 1700RCF, followed by mononuclear cell layer isolation and suspension in 10 ml of PBS. We isolated CD4+ lymphocytes using anti-CD4+ microbeads by column separation (Miltenyi Biotec, Auburn, CA) using 20 µl anti-CD4+ Micro beads per 106 total cells
2010-06-23 | E-GEOD-22324 | biostudies-arrayexpress
Project description:Serum were obtained from 4 groups, day0 and day28 neonates with CLD and non-CLD. Serum samples (250ul) from cord blood at birth and venous blood of neonates at 28 DAYS were transferred to sterile tubes containing 63 μl of ExoQuick precipitation solution (System Bioscience, Mountain View, CA, USA), and mixed. The mixtures were incubated for 30 min at 4℃, and centrifuged at 1500×g for 30 min. Then, the supernatants were removed, the pellets (extracellular vesicles) were diluted to 60 μl with phosphosaline buffer, and then thawed on dry ice for total RNA extraction with a miRvana isolation kit (Ambion). 3pM synthetic ath-miR159a was spiked into each sample to correct for technical differences. Using a human miRNA array, we identified 62 miRNAs that were universally expressed in CLD patients and non-CLD patients (Normalized Template).
2021-04-06 | GSE129400 | GEO
Project description:Three breast cancer cell lines were co-cultured with T cells isolated from normal donors, in 3: 1 ratio. As controls, the breast cancer cells were cultured without T cells for three days. All cell cultures were washed three times with PBS to remove T cells before they were scraped and collected in 1.5 mL Eppendorf tubes. Three additional washing steps with PBS were applied. After removing the supernatant, the cell pellets were immediately snap-frozen on dry-ice and stored in -80 °C until the time of preparation. After thawing the samples, they were prepared and measured on nano LC.
2018-01-24 | PXD006750 | Pride
Project description:The aim of the experiment was the identification of genes that are expressed in the apical organ of the sea anemone Nematostella vectensis. To achieve this, animals were generated that either lack the apical organ (injected with a morpholino antisense oligonucleotide to block the function of NvFGFa1) or have an expanded apical organ (injected with a morpholino antisense oligonucleotide to block the function of NvFGFa2, see Rentzsch et al., Development, 2008). Eggs from the same batch were injected after fertilization, (MO NvFGFa1 at 0.5uM, MO NvFGFa2 at 0.25uM) and planula larvae with the expected phenotype and without general developmental abnormalities were selected after 72 hours of development at room temperature (22°C), and placed in RNAlater Solution (Ambion) for subsequent extraction of RNA. Three conditions were isolated: embryos injected with NvFGFa1 MO, embryos injected with NvFGFa2 MO and non-injected embryos. Total RNA was extracted with the RNAqueous kit (Ambion), following the manufacturer’s protocol, and temporarily stored at -80°C, in order to accumulate the quantity necessary for the hybridization. The isolated total RNA for each condition was pooled and split into to two tubes and shipped on dry ice to Nimblegen in Iceland. The injections and the isolation of total RNA were performed from May – August 2009 at the Sars Centre in Bergen, Norway.
2014-10-19 | E-MTAB-3004 | biostudies-arrayexpress
Project description:100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. Keywords: stress response, dose response
2008-01-03 | GSE7609 | GEO
Project description:Transcript profiles of Phanerochaete chrysosporium grown on ball-milled aspen or ball-milled pine were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignocellulose degradation. From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and M-BM-< volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20M-KM-^ZC. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturerM-bM-^@M-^Ys protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20M-KM-^ZC. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42M-KM-^ZC. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16M-KM-^ZC for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16M-KM-^ZC. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37M-KM-^ZC. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37M-KM-^ZC. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42M-KM-^ZC. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.
2011-06-02 | E-GEOD-27941 | biostudies-arrayexpress
Project description:Tumor neurospheres were grown in culture until 90% confluent. To collect cells, cells were filtered through 0.45um nylon filters (Fischer Sci 099029) that were prewashed with methanol and ultrapure water using a microanalytical glass filter holder (Fisher Sci 09753E). The cells were filtered through filtration apparatus, then washed with 1mL of distilled water very quickly. Filter paper is taken immediatedly with tweezers and plunged cell side down into 6-well plate filled with liquid nitrogen placed on dry ice. Plate is wrapped in aluminum foil and stored at -80C before the liquid nitrogen evaporates. Samples are shipped on dry ice
2015-06-10 | ST000199 | MetabolomicsWorkbench
Project description:To determine the effect of RNA degradation on miRNA expression profiling. To mimic total RNA degradation in snap-frozen livers and duodenums from mice (wild type female, C57/Bl6) tissues were stored in a -80°C freezer and then transferred to dry ice. Each frozen tissue was sliced into five identical pieces which then were transferred into eppendorf tubes. At time point zero (T0), all samples were placed on ice and total RNA was extracted immediately from (T0) and at later time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA integrities were assessed using the Agilent Bioanalyzer 2100 which calculates RIN values of assayed RNAs. We found that at T0, the RNA integrity for both liver and duodenum was above 7, indicating good quality total RNA. However, 30 min on ice was sufficient to reduce RNA integrity, indicated by the decrease in RINs. These findings indicate that RNA degradation can take place in defrosted tissue at low temperature (i.e. on ice). For comparison, we assessed the extent to which RNA integrity is preserved in freshly harvested tissues when tissue processing is delayed. Liver and duodenum samples were collected from mice and either processed immediately or maintained on ice, as described above. Bioanalyzer electropherograms of the liver samples did not indicate any significant total RNA degradation even when samples remained on ice for up to 4 hrs. By contrast, duodenal samples (which are rich in RNases) do show high susceptibility to degradation similar to the snap-frozen defrosted material. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually. To assess to which extent total RNA degradation affects miRNA expression profiles, we hybridized to the miCHIP microarray total RNAs extracted from freshly harvested livers and duodenums liver and from snap frozen livers and duodenums. Hierarchical clustering (HCL), by Pearson correlation, was used to cluster samples based on their miRNA expression profiles. Our analysis shows that miRNAs extracted from freshly harvested liver is less degraded (matrix plot analyses). Consistent with high level of RNases present in duodenum, miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.
2010-05-19 | E-GEOD-19104 | biostudies-arrayexpress
Project description:Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Kidneys were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC (PMID: 30352994). The proteins captured by eRIC were separated by gel electrophoresis, divided into 7 fractions of defined mass, and identified by MS to determine their distribution across the fractions. Crosslinking to other proteins should shift the signal to higher mass fractions. The analyses were conducted in triplicate.
2023-03-02 | PXD038100 | Pride
Project description:Large White and Meishan pigs were either non-treated or injected with mammalian 1-24 ACTH (Immediate Synachten, Novartis France) at the dose of 250 µg per animal. Pigs were sacrificed either immediately after capture from their home cage (non-treated animals) or 1 hour following ACTH injection. Adrenal glands were immediately collected from pigs and frozen on dry ice and then stored at -80°C until RNA isolation. Keywords: stress response, adrenal, gene expression, pig
2008-01-07 | GSE8377 | GEO