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Sample GSM322623 Query DataSets for GSM322623
Status Public on Sep 20, 2008
Title Chondrocytes stimulated with methotrexate treated RASF rep 1
Sample type RNA
 
Source name Human chondrocytes isolated from lateral condyle of femur bone in culture from donor pool 10, stimulated with supernatant from methotrexate treated RASF
Organism Homo sapiens
Characteristics Human chondrocytes cultured in RPMI 1640 medium supplemented with 10% human serum, 100ng/ml amphotericin B, 100U/ml penicillin and 100µg/ml streptomycin for 2 passages, 3D cultured for 14 days in alginate beads in presence of 170µM L-ascorbic acid 2-phosphate and subsequently stimulated for 48h with supernatant of RASF cultured in the same medium and treated with methotrexate for 48h
Treatment protocol Human chondrocytes were isolated by enzymatic digestion (1 U collagenase P, 333 U collagenase II and 33 U hyaluronidase), passage 2 chondrocytes were encapsulated in alginate beads (20 Mio cells/ml in 1,5% (w/v) alginate) and stimulated with conditioned medium of NDSF, RASF or treated RASF
Growth protocol Human chondrocytes obtained from lateral condyle of femur bones of 6 donors post mortem were amplified by cultivation in RPMI 1640 medium supplemented with 10% human serum, 100ng/ml amphotericin B, 100U/ml penicillin and 100µg/ml streptomycin for 2 passages, subsequently 3D cultured in alginate beads for 14 days in presence of 170µM L-ascorbic acid 2-phosphate and finally stimulated with conditioned medium of RASF, NDSF, or treated RASF
Extracted molecule total RNA
Extraction protocol Prior to RNA extraction, alginate beads were solubilized on ice in 55mM sodium citrate, 30mM EDTA and 150mM NaCl and cells were centrifuged at 800g for 5min (4°C). Total RNA isolation was proceeded according to the manufacturer’s protocol. Additionally, a proteinase K and DNase I digestion were performed. Isolation of total RNA was done for the different stimulated donor chondrocytes separately. Afterwards, equal amounts of total RNA from three stimulated donor chondrocytes were pooled, yielding different experimental groups of chondrocytes stimulated with supernatant of NDSF, RASF or treated RASF
Label biotin
Label protocol 2.5 µg of total RNA was amplified and labeled using the GeneChip® one-cycle target labeling and control reagents (Affymetrix)
 
Hybridization protocol 50µg/ml of labeled cRNA was hybridized at 45°C for 16h using the Affymetrix GeneChip hybridization oven. Washing and staining was performed on the Affymetrix Fluidics 450 using the EukGE-WS2v4 protocol.
Scan protocol GeneChips were scanned using the Affymetrix GeneChip Scanner 3000
Description Gene expression data of human chondrocytes stimulated with supernatant of RASF treated for 48h with methotrexate
Data processing Data were processed with GCOS 1.4 (TGT = 150) and subsequently imported and further analyzed in the BioRetis online database (www.bioretis.de). Additionally, expression data was processed with Robust Multi-Array Analysis version 0.4a7 (RMA)
 
Submission date Sep 19, 2008
Last update date Sep 19, 2008
Contact name Thomas Häupl
E-mail(s) thomas.haeupl@charite.de
Phone +49 30 450513293
Organization name Charité
Department Rheumatology
Street address Charitéplatz 1
City Berlin
ZIP/Postal code 10117
Country Germany
 
Platform ID GPL96
Series (1)
GSE12860 Antirheumatic Drug Response in Human Chondrocytes: Potential Molecular Targets to Stimulate Cartilage Regeneration

Data table header descriptions
ID_REF
VALUE RMA log2 transformed signals

Data table
ID_REF VALUE
1007_s_at 7.182065882
1053_at 5.333581227
117_at 5.453792671
121_at 8.372059038
1255_g_at 4.60513566
1294_at 6.450642218
1316_at 5.192362438
1320_at 5.122917849
1405_i_at 4.736096033
1431_at 4.523972684
1438_at 6.01212506
1487_at 6.866946915
1494_f_at 6.428958966
1598_g_at 9.896840249
160020_at 10.28387745
1729_at 6.850784073
177_at 6.069153202
1773_at 5.931470986
179_at 8.858868817
1861_at 5.843873418

Total number of rows: 22283

Table truncated, full table size 496 Kbytes.




Supplementary file Size Download File type/resource
GSM322623.CEL.gz 2.2 Mb (ftp)(http) CEL
GSM322623.EXP.gz 508 b (ftp)(http) EXP
GSM322623_MAS5_data.txt.gz 145.7 Kb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table
Processed data provided as supplementary file

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