Comment[ArrayExpressAccession] E-GEOD-41038 MAGE-TAB Version 1.1 Public Release Date 2012-12-01 Investigation Title Ankylosing spondylitis vs control synovial biopsies Comment[Submitted Name] Ankylosing spondylitis vs control synovial biopsies Experiment Description We have compared synovial biopsies from ankylosing spondylitis and undifferentiated spondylitis patients with healthy controls and osteoarthritis patients Objective: In spondylarthropies, whole-genome gene expression profiling studies have been limited to peripheral blood to date. By undertaking a study in knee synovial biopsies from spondylarthropy (SpA) and ankylosing spondylitis (AS) patients we aimed to identified joint-specific candidate genes and pathways. These pathways may mediate systemic inflammation driven joint damaging processes and more specifically, the osteoproliferation that is characteristic of these conditions. Methods: RNA was extracted from six seronegative SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: 416 differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, osteoblast activity, B-cell associated, matrix catabolic, and metabolic pathways. The inflammatory mediator, MMP3, was strongly upregulated in AS/SpA samples and the Wnt pathway inhibitors DKK3 and Kremen1 were downregulated. Conclusion: Pathways mediating both systemic inflammation as well as local tissue changes were identified. This suggests initial systemic inflammation in spondylarthropies transfers to and persists in the local joint environment, subsequently mediating changes in genes directly involved in the destructive tissue remodelling. Fifteen knee synovial biopsy tissue samples consisting of six seronegative spondyloarthropy (SpA), two ankylosing spondylitis (AS), three osteoarthritis (OA) and four normal control biopsies were obtained from the Synovial Tissue Bank at the Repatriation General Hospital in Adelaide, South Australia with the appropriate ethical approval (Supplementary Table 1). All patients provided informed written consent. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Thomas Thomas Person First Name Gethin Gethin Person Email gethin.thomas@uq.edu.au Person Affiliation University of Queensland Diamantina Institute Person Phone +61 7 3176 2755 Person Fax +61 7 3176 5946 Person Address University of Queensland Diamantina Institute, Princess Alexandra Hospital, Woolloongabba, QLD, Australia Person Roles submitter Protocol Name P-GSE41038-1 P-GSE41038-5 P-GSE41038-4 P-GSE41038-2 P-GSE41038-3 P-GSE41038-6 Protocol Description ID_REF = VALUE = transformed by variance stabilization transformation (VST) then normalized by robust spline normalization (RSN) Standard Illumina scanning protocol Standard Illumina hybridization protocol biopsy collection: Fifteen knee synovial biopsy tissue samples consisting of six seronegative spondyloarthropy (SpA), two ankylosing spondylitis (AS), three osteoarthritis (OA) and four normal control biopsies were obtained from the Synovial Tissue Bank at the Repatriation General Hospital in Adelaide, South Australia with the appropriate ethical approval (Supplementary Table 1). All patients provided informed written consent. RNA was extracted from the formaldehyde-fixed paraffin-embedded (FFPE) tissue blocks using the Arcturus ParadiseM-) Plus Reagent System (Molecular Devices, Sunnyvale, CA) as per the manufacturerM-bM-^@M-^Ys protocol. 200ng of RNA was used in the Illumina Whole-Genome DASLM-. (cDNA-mediated Annealing, Selection, Extension, and Ligation) Gene Expression Assay according to the Illumina protocol. This technique has been specifically developed for whole-genome expression profiling of degraded RNA samples from archived tissue biopsies. This protocol has been specifically developed for whole-genome expression profiling of degraded RNA samples from archived tissue biopsies. RNA is first converted to cDNA through a reverse transcription reaction with biotinylated primers. The biotinylated cDNA is then annealed to assay oligonucleotide probes specific for each of the 24000 cDNAs targeted by the array. The hybridized oligonucleotides are then extended and ligated in a second-strand cDNA synthesis forming a synthetic template that is transferred to a PCR reaction containing a fluorescently labelled primer. The labelled PCR product strand is then isolated and the fluorescent products were hybridised to Illumina Ref-8 Expression BeadChips and scanned. Array data were processed using the Illumina BeadStudio software then the processed data were assessed for quality control and normalised in Lumi. Analysis of gene expression patterns was performed in BRB-ArrayTools. For quality control scanned images of the arrays were visually inspected for artefacts in Illumina BeadStudio followed by the graphical analysis of density plots in Lumi. The microarray data were transformed by variance stabilization transformation (VST) then normalized by robust spline normalization (RSN). Post-normalization quality control was carried out using density plots to ensure all samples had similar distribution and variance. To reduce background noise in the data analysis all probes that were not expressed in any of the samples, i.e. whose intensity was below background in all samples, were excluded from the analysis. This did not exclude probes for genes whose expression was either M-bM-^@M-^\switched-onM-bM-^@M-^] or M-bM-^@M-^\switched-offM-bM-^@M-^]. Protocol Type bioassay_data_transformation image_aquisition hybridization nucleic_acid_extraction labeling feature_extraction Experimental Factor Name BIOPSY Experimental Factor Type biopsy Comment[SecondaryAccession] GSE41038 Comment[GEOReleaseDate] 2012-12-01 Comment[ArrayExpressSubmissionDate] 2012-09-20 Comment[GEOLastUpdateDate] 2012-12-18 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE41038_non-normalized.txt SDRF File E-GEOD-41038.sdrf.txt