Project description:This series represents the effects of OVA, tg-IL-13, and direct effects of IL-13 on airway epithelial cells. Experiment Design: Type of experiment: comparison of three related murine models of asthma, 1) Ova model, 2) tg-IL-13 model, 3) IL-13/Epi model. Two control groups were used: PBS challenged mice to control for the effects of OVA and tg-IL-13+ and STAT6-/- mice as controls for tg-IL-13 and IL-13/Epi mice. Measurements were made in each model in two types of tissue samples, whole lung and tracheal perfusate. Experimental factors: Allergen vs sham challenge, natural STAT6 expression vs transgenic expression and no expression. The number of hybridizations performed in the experiment: 50 (25 whole lung and 25 tracheal perfusate). The type of reference used for the hybridizations, if any: Pooled reference from lung (whole lung or tracheal perfusate) from untreated wildtype mice. Hybridization design: two-color hybridizations with reference design. Each individual sample was hybridized to a separate array. Quality control steps taken: 5 experimental replicates per group. RNA integrity assessed by Agilent Bioanalyzer. Arrays with low signals, high background, or high spatial variation rejected and corresponding samples reanalyzed. URL of any supplemental websites or database accession numbers:GEO (http://www.ncbi.nlm.nih.gov/geo, accession number GSE1438). ********** Samples used, extract preparation and labeling: The origin of the biological sample: homogenized whole lung from mouse and perfusate of mouse trachea. Manipulation of biological samples and protocols used: Whole lungs mechanically homogenized in 7.0 ml Trizol reagent (Invitrogen) for total RNA collection. Tracheas perfused with lysis buffer (Qiagen Rneasy kit) for total RNA collection. Protocol for preparing the hybridization extract and labeling: Preparation of aminoallyl-UTP labeled whole lung cDNA was performed as described [1]. Total RNA from tracheal perfusate (1.0 - 1.5 g per sample) was amplified and labeled with aminoallyl-dUTP using the MessageAmp aRNA kit (Ambion). Labeled cRNAs were coupled to Cy3 or Cy5 dyes (CyScribe, Amersham Biosciences) and purified as described [1]. Labeling protocol(s): Coupled to Cy3 or Cy5.External controls (spikes): none. ********** Hybridization procedures and parameters: Hybridizations were performed as described [1] except Ambion SlideHyb Glass Array Hybridization Buffer #1 (neat concentration) was used as hybridization buffer and hybridizations were carried out for 40 h at 55 Â°C. Following hybridization, arrays were washed in 1X SSC with 0.03% SDS (wash 1), 0.2X SSC (wash 2), and 0.05X SSC (wash 3) for five minutes for each wash. ********** Measurement data and specifications: Arrays were scanned using an Axon Genepix 4000B scanner and GenePix Pro Analysis 5.0 software; laser power 100%, 10 micron resolution, PMT optimized for each array. Data from GPR files are available from GEO (http://www.ncbi.nlm.nih.gov/geo, accession number GSE1438).The âprint-tip loessâ normalization was used to correct for within-array dye and spatial effects and single channel quantile normalization was used to facilitate comparison between arrays. No background subtraction was performed. We used functions in the library marrayNorm of the R / Bioconductor package to perform these normalizations. After normalization we determined the log2 ratio of experimental sample intensity to reference sample intensity for each probe on each array. ********** Array Design: General array design Array design name: UCSF 10Mm Mouse v.2 Oligo Array (GEO GPL1089) and UCSF Gladstone 18K Mouse v.2 Oligo Array (GPL1196) Platform type: spotted oligonucleotides (70mers) Surface and coating specification: aminosilane coated glass slides Physical dimensions of array support (e.g. of slide): 25 x 75 x 1.0 mm Number of features on the array: GPL1089: 19152, GPL1196: 18240 (including empty and duplicate features) For production protocol, see http://www.microarrays.org/pdfs/PrintingArrays.pdf Feature information is available from GEO (GSE1438). Reporters: synthetic single stranded oligonucleotides. Sequence and annotation information available from GEO (GPL1089 and GPL1196) Method of reporter preparation: synthesized by Operon. The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking: see http://www.microarrays.org/pdfs/PrintingArrays.pdfAny additional treatment performed prior to hybridization: none. ********** Reference: 1. Barczak A, Rodriguez MW, Hanspers K, Koth LL, Tai YC, et al. (2003) Spotted long oligonucleotide arrays for human gene expression analysis. Genome Res 13: 1775-1785. Keywords = IL-13 Keywords = Epithelial Keywords = differential gene expression Keywords = microarray

Project description:NOD mice (8- to 13-wk-old) were bred and maintained at the La Trobe University Central Animal House (Bundoora, Melbourne, Australia). All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council 1997), after approval by the La Trobe University Animal Ethics committee (Melbourne, Australia). A total of 124 female mice were divided into three groups. The immunized group comprised 91 mice injected s.c. into the lower flanks with 200 micrograms of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The 26 mice in the control group were injected with adjuvant and pertussis toxin only. The seven animals used for the baseline group (time, day 0) were naive, not injected mice. Except for the seven naive animals from the baseline group, nine animals (seven from the EAE and two from the control group) were sacrificed (at the same time of the day) at each of 13 time points after immunization, which are detailed as days 3, 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 postimmunization. Lymph nodes from the remaining four animals of the immunized and from the two control groups were dissected, immersed in RNAlater (Ambion), and frozen at 20C. Lymph nodes were removed from RNAlater and homogenized in TRIzol (Invitrogen Life Technologies) using an electric homogenizer. After resuspending the final RNA pellet in water, samples were repurified using the RNeasy kit (Qiagen). cDNA was synthesized from 15 micrograms of total RNA using Superscript II RT (Invitrogen Life Technologies) and a modified dNTP mix containing dUTP. Samples were hydrolyzed by adding 10 microliters of 0.1 N NaOH, neutralized with 25 microliters of 1 M HEPES, and precipitated with 3 M sodium acetate and ethanol. Resuspension in 0.05 M sodium bicarbonate was followed by 1 h incubation with either N-hydroxysuccinimide ester Cy3 or Cy5 fluorescent dyes (Amersham Biosciences). Probes were quenched by the addition of 4 M hydroxylamine and neutralized with 100 mM sodium acetate. Final probe cleanup was conducted using the QIA Quick PCR purification kit (Qiagen). We followed a common reference design in which each Cy3-labeled lymph node probe was combined with a Cy5-labeled probe derived from a pool of RNA. Hybridization onto glass slides containing 18,240 spotted 60- to 70-mer oligonucleotides, followed by washing and scanning was performed at the Gladstone microarray core facility at the University of California (San Francisco, CA) Keywords: Time series NOD mice (8- to 13-wk-old) were injected s.c. into the lower flanks with 200 micrograms of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The mice in the control group were injected with adjuvant and pertussis toxin only. Disease and control mice were sacrificed at 13 identical timepoints and microarrays were processed for each timepoint.

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A.thaliana - Cotyledon, SC (Baerenfaller,integrbiol,2011)

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