Transcription profiling by array of Pseudomonas aeruginosa in a rhizopsphere during phosphate limitation conditions
ABSTRACT: Plants and rhizosphere microbes rely closely on each other, with plants supplying carbon to bacteria in root exudates, and bacteria mobilizing soil-bound phosphate for plant nutrition. When the phosphate supply becomes limiting for plant growth, the composition of root exudation changes, affecting rhizosphere microbial communities and microbially-mediated nutrient fluxes. To evaluate how plant phosphate deprivation affects rhizosphere bacteria, Lolium perenne seedlings were root-inoculated with Pseudomonas aeruginosa 7NR, and grown in axenic microcosms under different phosphate regimes (330 uM vs 3-6 uM phosphate). The effect of biological nutrient limitation was examined by DNA microarray studies of rhizobacterial gene expression.
Project description:Phosphate is essential for all living organisms, participating in critical biochemical processes, being an essential component of the energy dynamics of cells and being a component of nucleic acids, phospholipids in membranes, and other biomolecules. Therefore, the ability to withstand conditions of phosphate starvation, making use of the available phosphate, is of great importance for cell survival. Microarrays were performed to study the transcriptional response of Pseudomonas aeruginosa under phosphate deficient conditions.
Project description:Samples used in the study originated from three UK sites: the Walton Centre for Neurology and Neurosurgery in Liverpool, the Salford Royal Hospital in Salford and the Southern General Hospital in Glasgow. We recruited patients with pharmacoresistant mesial temporal lobe epilepsy for whom a therapeutic temporal lobectomy was being undertaken. After surgery, the hippocampus was divided into two portions: (1) one portion was preserved for RNA isolation, and (2) the other portion underwent histological analysis by an experienced neuropathologist. Frozen post-mortem histologically-normal hippocampal samples from donors with no known brain diseases were obtained from the MRC Edinburgh Brain Bank (Edinburgh, UK) and the Queen Square Brain Bank (London, UK). Brain samples were disrupted and homogenized in an appropriate volume of QIAzol lysis reagent (Qiagen, Crawley, United Kingdom) by using a TissueRuptor handheld rotor-stator homogenizer (Qiagen, Crawley, United Kingdom). Total RNA was extracted from the homogenates using the RNeasy Lipid Tissue Mini Kit (Qiagen, Crawley, United Kingdom), according to the manufacturer’s instructions. RNA quality was examined by capillary electrophoresis on an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA) and Agilent 2100 Expert software was used to calculate the RNA Integrity number (RIN) of each sample. Purity of the RNA sample was assessed using a NanoDrop1000 Spectrophotometer. Capillary electrophoresis traces were also examined. Samples with RNA integrity number scores (RIN) below 6, obvious RNA degradation, significant 18S or 28S ribosomal RNA degradation, ratio of absorbance at 260nm and 280nm <1.95, or with noticeable DNA or background contaminants did not pass QC, and were withheld from microarray analysis. The microarrays were processed at the Centre for Genomics Research in the University of Liverpool (http://www.liv.ac.uk/genomic-research/). 50ng of total RNA was amplified and labelled using the Agilent Low Input Quick Amp One-Colour Labeling Kit and labelled RNA was hybridized to Agilent SurePrint G3 Custom Exon 8x60K Microarrays designed to contain probes for each exon of 936 selected genes, including all known SLC genes. Standard Agilent protocols were followed. One array failed on five of the QC criteria and, hence, was excluded. Intensity data were extracted from the remaining arrays using the Feature Extraction Software, in line with the manufacturer’s recommendations. The uploaded files contain data both for a custom exon array designed to contain probes for each exon of 936 selected genes and for a custom gene expression array designed to contain gene expression probes for genes across the whole genome.
Project description:A murine model that mimic the decidualization and regression observed in human was used to investigate the molecular mechanisms underlying the dynamic processes in endometrium. Ovariectomized mice were treated sequentially with steroid hormones and then, to induce decidualization, oil was injected into the uterine lumen. A process similar to menstruation was induced by hormone-withdrawal. The uterine tissues were collected at 4 time-points after the induction of decidualization. Experiment Overall Design: The uterine tissues were collected at 36 (T1), 48 (T2), 60 (T3) and 84 hours (T4) after the last progesterone injection. There are 4 experimental animals for each time-point, and the RNAs collected from animals within the same time-point group were pooled together for Affymetrix microarray analysis using U74Av2 Chips.
Project description:RNAi-mediated silencing of Rab5 and the consequent loss of endosomes in the mouse liver caused severe metabolic defects such as hypoglycemia, hepatomegaly, hypercholesterolemia, hyperlipidemia and glycogen accumulation. Gene expression in mice liver after knockdown of Rab5 has been performed the Mouse Genome 430 2.0 microarrays to identify biological processes and pathways affected by Rab5. The mice received either PBS or lipid nanoparticles (LNPs) loaded with siRNAs agains Rab5all or Luciferase control at 1.5 mg/kg via tail vein injection. At 5 day post injection mice were sacrificed using cervical dislocation and liver tissue was harvested, snap frozen in liquid nitrogen for RNA isolation; Gene expression studies were performed applying the Mouse Genome 430 2.0 microarrays.
Project description:Comparison of whole genome and tiled gene expression between different colour pattern races of H. melpomene in developing wings. A total of 96 ds-cDNA samples labelled with Cy3 were hybridized onto 8 custom-designed Roche NimbleGen 12x135K format microarrays (Roche NimbleGen) for a total of 96 identical subarrays with 135,000 probes of 60 bp each and each hybridising a separate sample. The number of samples corresponded to two races, four stages and four tissue sections (forewing proximal, forewing distal, hindwing and eye), all of them with three independent replicates.
Project description:The expression of K-ras transgene is induced in K-rasV12+/-/Cre+/+/ROSA26R-LacZ+/+ transgenic mice uteri, which were primed with hormone treatment. Decidualization was artificially induced in these uteri and hormone injection was withdrawn to induced menstruation-like endometrial degeneration. Microarray was then used to study the molecular changes in these menstruating uteri following the K-ras activation. Uterine tissues were collected from K-ras induced and littermate controled menstruating uteri. Experiments were carried out using Illumina Mouse WG-6 BeadChips (n=6 in each group). Data were normalized in R environment using the Lumi Bioconductor package.
Project description:Early, low risk IPSS (International Prognostic Scoring System) myelodysplasia (MDS) is a heterogeneous disorder where the molecular and cellular haematopoietic defects are poorly understood. To gain insight into this condition, we analyzed gene expression profiles of marrow CD34+ progenitor cells from normal karyotype, low blast count MDS patients, age-matched controls and patients with non-MDS anaemia. The aim of the study was to further understanding of the cellular defect in MDS and to identify biomarkers of disease Experiment Overall Design: Bone marrow (BM) CD34 cells were purified from patients with MDS, non-MDS anemia and from normal donors. Total RNA was extracted from Tri-reagent and quality verified on by capillary electrophoresis (Agilent). RNA was amplified by the Affymetrix small sample protocol. cRNA was hybridised to Affymetrix U133A chips under standard conditions. Initial data was analysed in MAS 5.0
Project description:Here we examined virulence activation in Pseudomonas aeruginosa in response to the synthetic kappa opioid agonist U-50, 488 in nutrient poor media where growth conditions are limited and density dependent quorum sensing is not activated. We used a microarray to define the virulence-related genes in P. aeruginosa grown in liquid poor nutrient medium to determine the effect of kappa-opioid receptor agonist on virulence/lethal phenotype All biological samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from liquid culture at 7 hrs of growth in 1.) 0.1xTY medium containing tryptone 1 g/L and yeast extract, 0.5 g/L, or 2.) 0.1xTY+25 mM potassium phosphate buffer, pH 6.0, or 3.) 0.1xTY+200 uM U-50,488, or 4.) 0.1xTY+ 25 mM potassium phosphate buffer, pH 6.0+ 200 uM U-50,488 were used for RNA isolation. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility
Project description:Whole-genome expression microarray experiments were conducted to assess the response of C. metallidurans CH34 to aqueous Au(III)-chloride and identify possible biochemical pathways for Au detoxification. Four microarray experiments were conducted to assess gold response at low, medium and high Au(III)-chloride concentrations (i.e., 10, 50 and 100 µM Au(III)-chloride) and different induction times (10 and 30 minutes with 50 µM Au(III)-chloride). Based on these arrays, the differentially expressed genes upon gold-exposure can be identified. The four microarray experiments (10, 50 and 100 µM Au(III)-chloride at 10 minutes and 50 µM Au(III)-chloride at 30 minutes) were all performed in biological triplicate and containing three (in-slide) technical repeats. For all conditions, the metal-induced sample (Cy5) was compared with the non-induced sample (Cy5) to identify those genes that were differentially expressed upon gold exposure.
Project description:P. aeruginosa PAO1 grown as lawns on Nematode Growth Medium prepared without supplementation (NGM Pi<0.1 mM) has high killing ability against C. elegans, however, no mortality in worms has been observed during 48 hrs when feeding on PAO1 lawns grown on phosphate supplemented full NGM Pi 25 mM, pH 6.0 medium. We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns in NGM Pi<0.1 mM vs NGM Pi25 mM pH 6.0 All samples for gene expression analysis were prepared in biological triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or NGM/Pi<0.1 mM were used for RNA isolation. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility