|
| Status |
Public on Dec 10, 2010 |
| Title |
MS121 AR H |
| Sample type |
RNA |
| |
|
| Source name |
RASF, hypoxia, 0.5% Ox, 22h
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Synovial fibroblasts culture
gender: Female
age: 72y
disease status: rheumatoid arthritis patient
|
| Treatment protocol |
Synovial tissues were obtained from 6 patients with RA at the time of knee prostetic replacement surgery and 6 sex and age matched adult donors without history of joint disease and macroscopically healthy joints either at necropsy or at elective knee arthroscopic surgery for meniscal tears. Fibroblast cultures were established by explant growth in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (Cambrex Bio Science, Verviers, Belgium). Cultures were used after passage 4 in order to avoid contamination by other cell types. Parallel subconfluent cultures were incubated for 22 hours under normoxic or hypoxic conditions in a cell incubator under a controlled (CO2/N2/O2) atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from synovial fibroblasts was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality control was performed in 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). In all cases, RIN (RNA integrity number) was higher than or equal to 9.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485050865_S01_GE1-v5_10_Apr08_1_2.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 014850_D_20070207) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 21, 2010 |
| Last update date |
Dec 10, 2010 |
| Contact name |
Manuel J Del Rey |
| E-mail(s) |
ermanu3@yahoo.es
|
| Phone |
0034 91 3908766
|
| Organization name |
Instituto de Investigación Hospital 12 de Octubre
|
| Department |
Grupo de Enfermedades Inflamatorias y Autoinmunes
|
| Street address |
Avda Córdoba s/n
|
| City |
Madrid |
| ZIP/Postal code |
20041 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE21959 |
Transcriptional response to hypoxia of normal and rheumatoid arthritis synovial fibroblasts |
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