title: Differential transcriptome analysis of intraarticular lesional vs intact cartilage reveals new candidate genes in osteoarthritis pathophysiology.
abstract: OBJECTIVE
  To elucidate disease-specific molecular changes in osteoarthritis (OA) by analyzing the differential gene expression profile of damaged vs intact cartilage areas within the same joint of patients with OA of the knee using a combination of a novel RNA extraction technique and whole-genome oligonucleotide arrays.
  
  
  METHODS
  The transcriptome of macroscopically affected vs intact articular cartilage as determined by visual assessment was analyzed using an optimized mill-based total RNA isolation directly from the tissue and high density synthetic oligonucleotide arrays. Articular cartilage samples were obtained from patients with OA of the knee. Expression of differentially regulated genes was validated by real-time quantitative polymerase chain reaction and immunohistochemistry.
  
  
  RESULTS
  The amount of RNA obtained by the optimized extraction procedure was at least 1 microg per 500 mg of cartilage and fulfilled the common quality requirements. After hybridization onto HG-U133 Plus 2.0 GeneChips (Affymetrix), 28.6-51.7% of the probe sets on the microarray showed a detectable signal above the signal threshold in the individual samples. A subset of 411 transcripts, which appeared to be differentially expressed, was obtained when applying predefined filtering criteria. Of these, six genes were found to be up-regulated in the affected cartilage of all patients, including insulin-like growth factor binding protein 3 (IGFBP-3), wnt-1-inducible signaling protein 1 (WISP-1), aquaporin 1 (AQP-1), delta/notch-like EGF-repeat containing transmembrane (DNER), decay accelerating factor (DAF), complement factor I (IF).
  
  
  CONCLUSIONS
  The optimized methodical approach reported here not only allows to determine area-specific gene expression profiles of intraindividually different low-RNA containing OA cartilage specimens. In addition, this study also revealed novel genes not yet reported to play a role in the pathophysiology of joint destruction in OA.
authors.affiliation: Department of Rheumatology and Clinical Immunology, Justus-Liebig-University Giessen, Kerckhoff-Klinik, Bad Nauheim, Germany. m.geyer@kerckhoff-klinik.de
authors.forename: M
authors.initials: M
authors.lastname: Geyer
authors.forename: S
authors.initials: S
authors.lastname: Grässel
authors.forename: R H
authors.initials: RH
authors.lastname: Straub
authors.forename: G
authors.initials: G
authors.lastname: Schett
authors.forename: R
authors.initials: R
authors.lastname: Dinser
authors.forename: J
authors.initials: J
authors.lastname: Grifka
authors.forename: S
authors.initials: S
authors.lastname: Gay
authors.forename: E
authors.initials: E
authors.lastname: Neumann
authors.forename: U
authors.initials: U
authors.lastname: Müller-Ladner
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D051398:Aquaporin 1 / Q000235:genetics; D055511:CCN Intercellular Signaling Proteins; D018958:CD55 Antigens / Q000235:genetics; D002358:Cartilage, Articular / Q000737:chemistry* / Q000473:pathology; D017244:Complement Factor I / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012313:RNA / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000235:genetics; D015854:Up-Regulation
chemical_list: D055511:CCN Intercellular Signaling Proteins; C116396:CCN4 protein, human; D018958:CD55 Antigens; C462501:DNER protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D051398:Aquaporin 1; D012313:RNA; D017244:Complement Factor I