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| Status |
Public on Dec 30, 2013 |
| Title |
Synovial tissue prior to Methotrexate therapy, repl 5 |
| Sample type |
RNA |
| |
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| Source name |
Knee synovial biopsies, obtained by needle arthroscopy, in untreated patient with early rheumatoid arthritis (< 1 year disease duration)
|
| Organism |
Homo sapiens |
| Characteristics |
gender: F
age: 53
disease activity score (das)-28crp: 2.89
|
| Treatment protocol |
All patients included in the protocol met the American College of Rheumatology 1987 revised classification criteria for RA. They were all methotrexate (MTX) naive at inclusion and 3 of them were treated with low-dose glucocorticoids (prednisone ≤ 7.5 mg/day). All patients presented knee synovitis at inclusion. Patients were randomized into 2 groups: 12 received intravenous 8 mg/kg/month tociliuzumab (TCZ) therapy during 6 months, and 8 were treated with MTX that was administered orally at a 15 mg/week dose. Disease activity at baseline and at 3 months was evaluated using Disease Activity Score (DAS)-28CRP.
Synovial biopsy samples from 20 patients (12 in TCZ-treated group, 8 in MTX-treated group) were obtained by needle mini-arthroscopy of the affected knee at baseline (T0) and 12 weeks (T12) after initiation of therapy. For each procedure, 4–8 synovial samples were kept overnight at 4°C in an RNA stabilizing solution (RNALater; Ambion) and then stored at –80°C for later RNA extraction.The same amount of tissue was snap-frozen in liquid nitrogen and kept at –80°C for immunostaining experiments on frozen sections. The remaining material was fixed in 10% formaldehyde and embedded in paraffin for conventional optical evaluation and immunostaining of selected markers. All the experiments (RNA extraction, histology, immunohistochemistry) were performed on at least 4 biopsy samples harvested during every procedure in order to correct for variations related to the potential heterogeneous distribution of synovial inflammation. The study was approved by the ethics committee of the Université catholique de Louvain, and informed consent was obtained from all patients.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the synovial biopsy samples using the Nucleospin® RNA II extraction kit (Macherey-Nagel, Düren, Germany), including DNase treatment of the samples. At least 2 µg total RNA could be extracted for further processing from 12 and 8 paired samples at T0 and T12 in TCZ- and MTX-treated groups, respectively. RNA quality was assessed using an Agilent 2100 Bioanalyzer and RNA nanochips (Agilent Technologies, Santa Clara, CA). All samples had a RNA integrity number (RIN) > 6.5
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| Label |
Biotin
|
| Label protocol |
Complementary RNA (cRNA) was synthetized and biotin-labeled according to a standard Affymetrix procedure (GeneChip® 3’ IVT express kit, Affymetrix, High Wycombe, UK), as previously described. Briefly, total RNA was first reverse transcribed into single-stranded complementary DNA (cDNA) using T7 oligo(dT) primer. The second-strand cDNA is then synthetized using DNA polymerase and Rnase H to simultaneously degrade RNA. The double-stranded cDNA served as template for the in vitro transcription reaction, which was performed for 16 hours in the presence of a biotinylated ribonucleotide analog mix. At the end of the procedure, biotinylated cRNA was purified and fragmented to prepare the target for hybridization to GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix, UK)
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| |
|
| Hybridization protocol |
GeneChip® HGU133 Plus 2.0 arrays were hybridized overnight at 45°C with 10 µg fragmented biotinylated cRNA. The slides were then washed and stained using EukGE-WS2v5 fluidics protocol on the GeneChip® Fluidics station 450.
|
| Scan protocol |
The slides were scanned on the GeneChip® Scanner 3000 (Affymetrix, UK).
|
| Description |
MTX5 T0
|
| Data processing |
Statistical analyses of microarray data were performed using GeneSpring software (Agilent Technologies). Fluorescence intensity data were normalized by robust multiarray analysis.
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| |
|
| Submission date |
Apr 08, 2013 |
| Last update date |
Dec 30, 2013 |
| Contact name |
Bernard Robert Lauwerys |
| E-mail(s) |
Bernard.Lauwerys@uclouvain.be
|
| Phone |
+3227645391
|
| Organization name |
Université catholique de Louvain
|
| Department |
Institut de Recherches Expérimentales et Cliniques
|
| Lab |
Pôle de pathologies rhumatismales et systémiques
|
| Street address |
Avenue Hippocrate 10
|
| City |
Brussels |
| ZIP/Postal code |
1200 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE45867 |
Effects of tocilizumab versus methotrexate therapy on gene expression profiles in the early rheumatoid arthrtis synovium |
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