Transcription profiling of mouse expressing human Cathepsin L inhibitor hurpin, skin alterations and increased carcinogenesis.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Birgit Andree
PROVIDER: E-GEOD-5236 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesis.
Walz Markus M Kellermann Sabine S Bylaite Matilda M Andrée Birgit B Rüther Ulrich U Paus Ralf R Kloepper Jennifer E JE Reifenberger Julia J Ruzicka Thomas T
Experimental dermatology 20070901 9
The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed in ...[more]
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Our results suggest that the colonic inflammation in EF and EF.CIF-inoculated IL-10-/- mice is mediated by mechanisms similar to those of human IBD, in particular CD. Example logFC calculation; This example describes the calculation of the fold-change value (logFC) for a single spot (in this case, spot 27222) for a single comparison. This calculation method was applied to generate all of the comparison data shown in the supplementary data files. The comparison in question is for:; colon tissue from C57BL/6J mouse with EF.CIF treatment (represented by duplicate samples GSM307282 (Title: C57BL/6J_Colon_EF.CIF_Rep1_(Pool17)) and GSM307283 (Title: C57BL/6J_Colon_EF.CIF_Rep2_(Pool18)); vs. colon tissue from C57BL/6J mouse under conventional conditions (represented by duplicate samples GSM307270 (Title: C57BL/6J_Colon_Conventional_Rep1_(Pool5)) and GSM307271 (Title: C57BL/6J_Colon_Conventional_Rep2_(Pool6)); Important Note: Limma analysis maps the treatments on each slide to the M values which are by convention always log2(R) - log2(G). Because this experiment is a reference design, with the reference RNA always Cy5 (i.e. red, channel 2) and the treatment sample Cy3 (i.e. green, channel 1), and we are interested in the values of the treatment samples compared with the reference, the M values in the data files are displayed as log2(G) - log2(R); The average expression is what is important here, which is calculated from the normalized M values for the appropriate comparison of interest as follows:; [1] Mean value for Spot 27222, for each treatment:; C57BL/6J mouse, EF.CIF: = mean (0.26363587 (GSM307282), 0.05084283 (GSM307283)) = 0.15723935 C57BL/6J mouse, conventional = mean (1.42951674 (GSM307270),1.15444106 (GSM307271)) = 1.2919789; [2] Calculation of LogFC for the comparison of interest:; The contrast of interest is C57BL/6J: EF.CIF vs Conventional, so by performing a subtraction of these values for the comparison of interest (i.e. (mean for EF.CIF) - (mean for Conventional), the common reference is cancelled out, thus:; C57BL/6J: EF.CIF vs Conventional = 0.15723935 - 1.2919789 = -1.13473955; So, the fold-change value (logFC) for spot 27222 for the comparison in question (C57BL/6J: EF.CIF vs. Conventional) is: -1.13473955 Experiment Overall Design: Animals. Twenty five male IL-10-/- (C57BL/6J background, formal designation B6.129P2-IL-10<tm1Cgn>/J) mice and twenty five male C57BL/6J (C57) control mice were received from The Jackson Laboratory (Bar Harbor, Maine, USA) at approximately five weeks of age. Mice were housed either in pairs or groups of three (5 mice per treatment) in shoebox-style cages containing untreated wood shavings and a plastic tube for environmental enrichment. The animal room was maintained at a temperature of ~22°C and humidity of ~50% with a 12 hour light/dark cycle. All mice had ad libitum access to water, which was refreshed twice a week. An AIN-76A powdered diet was supplied twice a week, with sufficient provided to meet the daily intake of IL-10-/- mice, as determined in a previous feeding trial (data not shown). 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The proximal half of the colon was cut in two pieces, one for histological evaluation and other for gene expression studies. Samples of the colon tissue for gene expression analysis were frozen in liquid nitrogen and stored at - 80 ºC. Samples for histological analysis were stored in formalin solution (10 % neutral buffered) at room temperature. Experiment Overall Design: Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: The Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., Palo Alto, California, USA) was used to synthesize cDNA and fluorescent cRNA. Labelled cRNA was made on the same day for all sample pools, including the reference sample. cDNA was synthesized from 500 ng of purified total RNA from each pool using T7 promotor primer and Moloney Murine Leukemia Virus Reverse Transcriptase according to the manufacturerâ??s protocol. Cy3 (PerkinElmer, Waltham, Massachusetts 02451, USA) was used to label sample groups, while the reference RNA was labeled with Cy5 (PerkinElmer, Waltham, Massachusetts 02451, USA). Experiment Overall Design: HYBRIDIZATION Experiment Overall Design: Microarray hybridization was performed according to a reference design without dye swap. The in situ hybridization kit-plus (Agilent Technologies Inc., Palo Alto, California, USA) was used to hybridize cRNA samples to Agilent Technologies Mouse G4121A - 44k 60mer oligonucleotide arrays. Cy3-labelled cRNA (sample, 0.75 µg) and Cy5-labelled cRNA (reference, 0.75 µg) were hybridised onto the microarray according to the manufacturerâ??s protocol. Following hybridisation, slides were washed in solutions I, II and III (Agilent Technologies, Santa Clara, CA, USA) and air-dried. Experiment Overall Design: SCAN PROTOCOL Experiment Overall Design: Slides were scanned using a GenePix 4200A scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) at a photomultiplier tube (PMT) setting of 450 V. Spot identification and quantification were performed using GenePix 6.0 software (Molecular Devices Corporation, Sunnyvale, CA, USA). Spot identification and quantification was performed using GenePix 6.0 software (Molecular Devices). All slides were individually checked and manually flagged for abnormalities. Experiment Overall Design: DATA PROCESSING Experiment Overall Design: Statistical analysis was performed using linear models for microarray analysis (limma) within the Bioconductor framework. Before analysis, poor quality spots were manually flagged and filtered out. Quality of the microarray data was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. All twenty arrays passed these strict criteria and were included in the analyses. Intensity ratio values for all microarray spots were normalized using a within-slide global Locally Weighted Scatterplot Smoothing (Loess) procedure to remove the effect of systematic variation in the microarrays; no background correction was necessary due to homogeneous hybridization. The normalized data from the arrays of each treatment group were averaged. For each comparison, differentially expressed genes were identified using false discovery rate (FDR) control with a threshold of q < 0.05. Data were log transformed before analysis and the mean difference between treatments calculated on this scale, resulting in a log ratio for each probe. The normalized values in the database consist of these log ratios. MA plots of the microarray data were drawn in order to check that there was no dependence of the log ratio on the intensity for any slide. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe using an empirical Bayes approach. Experiment Overall Design: The probability values were then corrected for multiple testing using the Benjamini and Hochberg correction, and a false discovery rate (FDR) calculated. Probes that had an FDR of less than 5 % (q<0*05) were considered to be differentially expressed between treatment.
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