Boxed Experiment of Renal, brain and lung macrophages
Ontology highlight
ABSTRACT: Three way boxed experiment comparing EGFP positive renal, lung and brain macrophages. Each "arm" of the comparison is a pairwise comparison containing a dye swap to eliminate dye bias.
Project description:Pairwise comparison of total kidney RNA extracted from Nas1 knockout mice versus total kidney RNA extracted from wildtype mice Pairwise comparison containing a dye swap to eliminate dye bias
Project description:Direct pairwise comparison of the differential expression patterns between embryos injected with Standard olignucleotide morpholinoes and those injected with an anti-klf4 morpholino. Keywords: Direct pairwise comparison Each "arm" of the comparison is a pairwise comparison containing a dye swap to eliminate dye bias.
Project description:Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). Targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, we discovered a 1.8kb region between HOXD11 and HOXD12 (D11.12) that is associated with PcG proteins, is nuclease hypersensitive, and shows alteration as hESCs differentiate. D11.12 repressed luciferase expression from a reporter construct both before and after differentiation of mesenchymal stem cells into adipocytes. Full repression by D11.12 required a highly conserved region and YY1 binding sites. Repression relied upon PcG proteins Bmi1 and Eed and a YY1-interacting partner, RYBP. We conclude that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies. Data contains microarray measurements of the MNase-digested mononucleosomal fragments in hES cells, derived MSCs, osteoblasts and adipocytes. The ChIP-chip data includes Suz12, Bmi1 and H3K27me3 measurements in MSCs and adipocytes.
Project description:we want to test how different diets (high energy diet HED and low energy diet LED) alter muscle methabolism in pigs. we perform different array experiments using an human platform (GPL2011) and RNA extracted from pig skelethal muscle. thank's this we also test cross-species hybridisation. extraction of RNA from pig skeletal muscle by a modification of Chomczynski protocol. cDNA was purified and labelled with Cy3 and Cy5 fluorochromes using the cDNA labeling purification module kit (Invitrogen). The HED samples were analysed in comparison to LED samples, used as reference. The labelled cDNA were appropriately coupled and used for competitive hybridisation on the same microarray at 42°C for 16h. The relative intensity of labelled cDNA in HED and LED was acquired with ScanArray LITE scanner (PerkinElmer Life Sciences, Inc). platform used for our experiments is GPL2011.
Project description:This SuperSeries is composed of the following subset Series: GSE9242: Dietary folate depletion and repletion in A/J and C57BL/6J mice GSE9243: Dietary folate depletion and repletion in C57BL/6J mice, ApoE knockout mice, and choline supplemented C57BL/6J mice Keywords: SuperSeries Refer to individual Series
Project description:Defects in homocysteine and folate metabolism are associated with increased risks for neural tube and congenital heart defects, cardiovascular disease and stroke, cancers, and neurodegeneration. In many but not all cases, dietary supplementation with folate significantly reduces the severity and incidence of these conditions. Common polymorphisms modulate these metabolic pathways and disease risks, but do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals. To test whether other pathways contribute to disease pathogenesis, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice. Compared to the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes. Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and translated to changes in levels of serum and liver total cholesterol. By genetically increasing serum and liver total cholesterol levels in APOE deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting an interplay between homocysteine and folate metabolism and cholesterol metabolism. Absence of measurable changes in global methylation patterns or amelioration of effects with supplementation with an alternative methyl donor suggest that dietary folate perturbations do not act through large-scale or general changes in methylation. These results suggest that homeostatic responses in cholesterol metabolism contribute to the beneficial effects of dietary folate supplementation. Keywords: time course, stress response, diet, genetic, homeostasis Six-week old female A/J and C57BL/6J mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Selected mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine. We had nine different treatment plans per strain with eight replicate mice per treatment. There were four folic acid depletion treatment in which mice were placed on folic acid deficient diet for 1, 2, 7, or 14 days. There were two folic acid repletion treatment in which mice were placed on folic acid deficient diet for 14 days followed by 1 day on control diet and another set of mice on 14 days of folic acid deficient diet followed by 7 days of control diet. There were three control time points in which mice were placed on the control diet for 0, 9, or 22 days. Eight biological replicate liver tissue from each treatment was pooled and total RNA from each pool and total RNA from Universal Mouse Reference RNA (Stratagene) were aminoallyl labeled with Cy3 and Cy5 in duplicate, with reversing of dyes.
Project description:Defects in homocysteine and folate metabolism are associated with increased risks for neural tube and congenital heart defects, cardiovascular disease and stroke, cancers, and neurodegeneration. In many but not all cases, dietary supplementation with folate significantly reduces the severity and incidence of these conditions. Common polymorphisms modulate these metabolic pathways and disease risks, but do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals. To test whether other pathways contribute to disease pathogenesis, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice. Compared to the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes. Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and translated to changes in levels of serum and liver total cholesterol. By genetically increasing serum and liver total cholesterol levels in APOE deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting an interplay between homocysteine and folate metabolism and cholesterol metabolism. Absence of measurable changes in global methylation patterns or amelioration of effects with supplementation with an alternative methyl donor suggest that dietary folate perturbations do not act through large-scale or general changes in methylation. These results suggest that homeostatic responses in cholesterol metabolism contribute to the beneficial effects of dietary folate supplementation. Keywords: time course, stress response, diet, genetic, homeostasis Six-week old female C57BL/6J and B6.129P2-Apoetm1Unc/J (Piedrahita et al. 1992) mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine, for 14 days. A subset of these mice were placed back on control diet for 7 days after 14 day depletion. There were three groups of mice that underwent folic acid depletion and repletion. ApoE knockout mice on C57BL/6J background, C57BL/6J mice supplemented with 25mM choline and 50mM saccharine in drinking water, and C57BL/6J mice supplemented with 50mM saccharine. Saccharine was used to reduce the bitter taste of choline in the drinking water. We also had C57BL/6J mice on the control diet for 14 days and 21 days. These mice served as controls for treated mice from each time point during hybridization. The folate level in the control diet for this study was significantly lower than the previous study (GSE9242) due to greater loss of folic acid by irradiation of the diet. There were eight replicate mice per treatment group per folic acid perturbation protocol. An equal amount (by weight) of liver tissue from eight replicate mice was separated into two pools of four replicate tissues each. Pooled RNA from treated mice and pooled RNA from control mice for each time point were aminoallyl labeled with Cy3 and Cy5 in duplicate with reversing of dyes.
Project description:Persistence of memory CD4+ T cells in ECs was coupled with the inactivation of FOXO3a transcriptional activities, which we have previously identified as a critical regulator of TCM survival. Indeed, expression levels of transcriptional targets of FOXO3a, endowed with pro-apoptotic and anti-proliferative functions, were lower in TCM and TEM from ECs as compared to ST individuals. Silencing the transcriptionally active form of FOXO3a by siRNA rescued TCM and TEM of STs from Fas-mediated apoptosis. Moreover the expression of FOXO3a dominant negative form (FOXO3a Nt) rescued the long-term survival of TCM from STs as these cells persisted as long as those derived from ECs. Overall, these studies indicate that FOXO3a activation is an important mediator of the shortened survival and heighteined turnover of TCM and TEM in chronic HIV infection. Targeting this pathway may provide a strategy to preserve memory T cell numbers in HIV infection. Keywords: comparative gene profile, cell-type comparison Isolation of CD4+T cell sub-populations. Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naive cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen). Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4C to avoid any changes in cell phenotype or gene expression. Sample RNA was extracted using an RNA extraction kit (Quiagen), then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer’s instructions. The amplified RNA (aRNA) was then verified for quality and quantity using the Agilent Bioanalyser and measuring the OD. Universal human RNA (Stratagene) was also prepared in the same way. Sample probes were prepared by direct labelling with 3 µg of the aRNA Cy-5 (R values) fluorescent dye while the universal RNA probes were prepared by direct labelling of universal aRNA with Cy-3 (G values). All patient samples were hybridized against amplified universal RNA at 37 ºC for 18h on a custom human Immune array. Detailed information on the labeling and hybridization procedures can be obtained at the Microarray Centre web page (University Health Network).
Project description:Genetically modified mice have been extensively used for analyzing the molecular events that occur during the tumor development. However, in many cases, if not all, it is uncertain to what extent the mouse models reproduce features observed in the corresponding human conditions. This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors. We used global gene expression patterns of 68 hepatocellular carcinoma (HCC) from 7 different mouse models and 91 human HCC from pre-defined subclasses to obtain direct comparison of the molecular features of mouse and human HCC. Total RNAs were isolated from frozen liver tissue using CsCl density gradient centrifugation methods. Total RNA from the livers of 10 wild type mice were pooled and used as reference in entire microarray experiments. To obtain gene expression profile data from four transgenic HCC mouse models, 20 μg of total RNAs from tissues were used to drive fluorescently (Cy-5 or Cy-3) labeled cDNA. At least two hybridizations were carried out for each tissue using dye-swap strategy to eliminate dye labeling bias.
Project description:E.coli K-12 W3110 was grown in LB medium and harvested at each time point. And time series microarray experiments were performed based on reference desgin. In reference design, the control sample is collected at one representative time point. Combining with data from sequential design, more acculate and reliable expression series could be collected. Keywords: Reference design The control sample is collected at one representative time point.