NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM609397 Query DataSets for GSM609397
Status Public on Oct 19, 2010
Title RTX11 T12
Sample type RNA
 
Source name Human synovial biopsies from the knee of a patients with RA prior to (T0) or after (T12) RTX therapy (EULAR Poor-responder).
Organism Homo sapiens
Characteristics treatment: 12 weeks of RTX therapy
gender: F
age (y): 47
sample type: synovial knee biopsy
disease state: rheumatoid arthritis
response: EULAR Poor-responder
tissue: synovium
Extracted molecule total RNA
Extraction protocol Samples stored at –80°C after overnight incubation in RNALater were used for the microarray hybridizations. Total RNA was extracted from the synovial biopsies using the Nucleospin® RNA II extraction kit (Macherey-Nagel), including DNase treatment of the samples. At least 1 µg total RNA could be extracted from 12 paired samples at T0 and T12 for further processing. RNA quality was assessed using an Agilent 2100 Bioanalyzer and RNA nanochips (Agilent technologies Inc).
Label Biotin
Label protocol Labeling of RNA (cRNA synthesis) was performed according to a standard Affymetrix® procedure (One-Cycle Target Labeling kit, Affymetrix UK Ltd., High Wycombe, UK); briefly total RNA was first reverse transcribed into single-stranded cDNA using a T7-Oligo(dT) Promoter Primer and Superscript II reverse transcriptase. Next, RNase H was added together with E.Coli DNA polymerase I and E. Coli DNA ligase, followed by a short incubation with T4 DNA polymerase in order to achieve synthesis of the second-strand cDNA. The purified double-stranded cDNA served as the template for the in vitro transcription reaction, which was carried overnight in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix. At the end of this procedure, the biotinylated complementary RNA (cRNA) was cleaned, and fragmented by a 35 -minute incubation at 95°C.
 
Hybridization protocol GeneChip® Human genome U133 Plus 2.0 Arrays (spotted with 1,300,000 oligonucleotides, grouped in 54,675 probe sets informative for about 47,000 transcripts originated from not less than 39,000 genes, Affymetrix UK Ltd, High Wycombe, UK) were hybridized overnight at 45°C in monoplicates with 10 µg cRNA.
Scan protocol The slides were then washed and stained using the EukGE-WS2v5 Fluidics protocol on the Genechip® Fluidics Station (Affymetrix) before being scanned on a Genechip® Scanner 3000.
Description twenty patients with ra (17 women and 3 men, average age +/- sem: 52,6+/-3,8 years) were included in the study. All patients met the American College of Rheumatology classification criteria for the diagnosis of RA. All patients had active disease at the time of tissue sampling and were resistant to TNF blockade. They all had erosive changes imaged on conventional x-rays of the hands and/or feet. All of them had a swollen knee at inclusion. Rituximab therapy was administrated at a dose of 1,000 mg IV at baseline (T0) and at week 2, together with 125 mg IV Methylprednisolone. Clinical parameters at baseline (T0) and 12 weeks after the initiation of therapy (T12) was evaluated using DAS(28)-CRP scores and clinical responses were assessed using EULAR response criteria. Synovial biopsies were obtained by needle-arthroscopy of an affected knee from all patients at T0 and T12. For each procedure, 4 to 8 synovial samples were kept overnight at 4°C in a RNA stabilizing solution (RNALater, Ambion, Applied Biosystems, TX, USA) 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 paraffin embedded for conventional optical evaluation and immunostaining of selected markers. All the experiments (RNA extraction, histology, immunohistochemistry) were performed on at least 4 biopsies 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.
Data processing For the initial normalization and analysis steps, data were retrieved on Affymetrix GCOS software. The frequency of positive genes (genes with a flag present) was between 40 and 50% on each slide. After scaling of all probe sets to a value of 100, the range of the reported amplification scales was between 0,8 and 6,7. The signals yielded by the poly-A RNA, hybridization and housekeeping controls (GADPDH 3’/5’ ratio < 2.3 in all the slides) were indicative of the good quality of the amplification and hybridization procedures.
 
Submission date Oct 18, 2010
Last update date Oct 18, 2010
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)
GSE24742 Effects of Rituximab on global gene expression profiles in the RA synovium

Data table header descriptions
ID_REF
VALUE MAS 5.0 signal
ABS_CALL Flag
DETECTION P-VALUE Detection p-value

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
AFFX-BioB-5_at 216.249 P 5.16732e-05
AFFX-BioB-M_at 376.077 P 4.42873e-05
AFFX-BioB-3_at 187.057 P 4.42873e-05
AFFX-BioC-5_at 719.518 P 4.42873e-05
AFFX-BioC-3_at 872.724 P 4.42873e-05
AFFX-BioDn-5_at 1847.9 P 4.42873e-05
AFFX-BioDn-3_at 3235.3 P 4.42873e-05
AFFX-CreX-5_at 9282.89 P 4.42873e-05
AFFX-CreX-3_at 11947.9 P 4.42873e-05
AFFX-DapX-5_at 285.48 P 4.42873e-05
AFFX-DapX-M_at 576.981 P 0.000146581
AFFX-DapX-3_at 936.952 P 4.42873e-05
AFFX-LysX-5_at 40.7797 P 0.000752926
AFFX-LysX-M_at 64.9822 P 0.000856509
AFFX-LysX-3_at 156.208 P 5.16732e-05
AFFX-PheX-5_at 48.1405 P 0.000146581
AFFX-PheX-M_at 120.233 P 0.00010954
AFFX-PheX-3_at 92.4621 P 7.00668e-05
AFFX-ThrX-5_at 58.769 P 0.002867
AFFX-ThrX-M_at 94.7272 P 5.16732e-05

Total number of rows: 54675

Table truncated, full table size 1637 Kbytes.




Supplementary file Size Download File type/resource
GSM609397_1_RTX11_T12.CHP.gz 301.6 Kb (ftp)(http) CHP
GSM609397_RTX11_T12.CEL.gz 4.3 Mb (ftp)(http) CEL
Processed data included within Sample table
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap
External link. Please review our privacy policy.