|
| Status |
Public on Apr 20, 2010 |
| Title |
RA-4 passage 1 vs. passage 7 b oncogene array |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rheumatoid arthritis synovial fibroblasts from Patient #4 (RA-4)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Rheumatoid arthritis synovial fibroblasts from Patient #4 (RA-4)
passage: passage 1
|
| Treatment protocol |
Culture conditions were kept constant during the experiments.
|
| Growth protocol |
Synovial tissues were obtained from synovial biopsies of RA patients undergoing joint surgery. Following enzymatic digestion, fibroblasts were seeded in culture flasks (= passage 0) and grown in DMEM containing 10% heat inactivated fetal calf serum and cultured at 37°C in 10% CO2. At 85-95% confluency, cells were passaged 1:2 and a part of the cells was harvested and total RNA was extracted and stored at -70°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted using the RNeasy spin column purification kit. To remove contaminating genomic DNA, total RNA was treated with DNase I (0.2 U/µl for 40 minutes at 37°C. RNA concentrations were measured, adjusted to 200 ng/µl and stored at -70°C.
|
| Label |
32P
|
| Label protocol |
RAP-PCR of total cellular RNA was performed using 250 ng RNA. First strand synthesis was carried out using MuLV reverse transcriptase (Promega) and 2 µM first strand arbitary primer. Second strand synthesis was performed using AmpliTaq Stoffel Fragment 2.8 µl [32P]dATP (3,000 Ci/mmol, 10 mCi/ml), and 4 µM arbitrary second primer. The reaction was cycled through 30 low stringency cycles 94°C, 35°C, 72°C). Primer combination: OPN23 (5'- CAG GGG CAC C-3') and OPN21 (5'-ACC AGG GGC A-3').
|
| |
|
| Channel 2 |
| Source name |
Rheumatoid arthritis synovial fibroblasts from Patient #4 (RA-4)
|
| Organism |
Homo sapiens |
| Characteristics |
passage: passage 7
tissue: Rheumatoid arthritis synovial fibroblasts from Patient #4 (RA-4)
|
| Growth protocol |
Synovial tissues were obtained from synovial biopsies of RA patients undergoing joint surgery. Following enzymatic digestion, fibroblasts were seeded in culture flasks (= passage 0) and grown in DMEM containing 10% heat inactivated fetal calf serum and cultured at 37°C in 10% CO2. At 85-95% confluency, cells were passaged 1:2 and a part of the cells was harvested and total RNA was extracted and stored at -70°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted using the RNeasy spin column purification kit. To remove contaminating genomic DNA, total RNA was treated with DNase I (0.2 U/µl for 40 minutes at 37°C. RNA concentrations were measured, adjusted to 200 ng/µl and stored at -70°C.
|
| Label |
32P
|
| Label protocol |
RAP-PCR of total cellular RNA was performed using 250 ng RNA. First strand synthesis was carried out using MuLV reverse transcriptase (Promega) and 2 µM first strand arbitary primer. Second strand synthesis was performed using AmpliTaq Stoffel Fragment 2.8 µl [32P]dATP (3,000 Ci/mmol, 10 mCi/ml), and 4 µM arbitrary second primer. The reaction was cycled through 30 low stringency cycles 94°C, 35°C, 72°C). Primer combination: OPN23 (5'- CAG GGG CAC C-3') and OPN21 (5'-ACC AGG GGC A-3').
|
| |
|
| |
| Hybridization protocol |
The PCR products were purified from unincorporated nucleotides and small cDNA fragments by column chromatography. The filters were prehybridized in prewarmed hybridization solution with 100 µg/ml fragmented denatured salmon sperm DNA. The labeled cDNA probe was denatured and 1 µg/µl sheared human genomic DNA was added with an equal volume of neutralizing solution (1M NaH2PO4, pH 7.0). The mixture was added to the filters and hybridized over night.
|
| Scan protocol |
The filters were exposed to a Phosphor-Imager-Screen for 3-5 days depending on the intensity of radiation. Data analysis was performed using the Ambis software.
|
| Description |
Array comparison evaluation using BD AtlasImageTM 2.7 Software providing data of differentially regulated genes (equally expressed genes are not provided by the software)
|
| Data processing |
Evaluation was performed using the AtlasImageTM 2.7 software, developed specifically for analysis of the AtlasTM cDNA Expression Arrays. Background and signal intensity was normalized. The default signal threshold was kept constant for all analyses. After background correction, the median signal intensity for all spots on an array were used to calculate the correction coefficient (global normalization). Comparison of the arrays was performed using the “Compare two arrays” setting and resulting .txt-files were saved as .xls-files. Genes spotted on both arrays were evaluated on the GLP139 array only.
*********Only differentially expressed genes are reported in the Sample data tables. Complete data tables were requested and reported to be unavailable by the submitter.*********
|
| |
|
| Submission date |
Apr 19, 2010 |
| Last update date |
Apr 19, 2010 |
| Contact name |
Elena Neumann |
| E-mail(s) |
e.neumann@kerckhoff-klinik.de
|
| Phone |
+4960329962801
|
| Fax |
+4960329962809
|
| Organization name |
Justus-Liebig-Universität Gießen, Kerckhoff-Klinik
|
| Department |
Internal Medicine and Rheumatolgy
|
| Lab |
Rheumatology Research
|
| Street address |
Benekestr. 2-8
|
| City |
Bad Nauheim |
| ZIP/Postal code |
61231 |
| Country |
Germany |
| |
|
| Platform ID |
GPL139 |
| Series (1) |
| GSE21385 |
Culture effects on rheumatoid arthrits synovial fibroblasts |
|
| Data table header descriptions |
| ID_REF |
|
| Intensity_1 |
Raw signal intensity Ch1 (Passage 1, reference) |
| Background_1 |
Background signal intensity Ch1 (Passage 1, reference) |
| Adj.Intensity_1 |
Normalized signal intensity Ch1 (Passage 1, reference) |
| Intensity_2 |
Raw signal intensity Ch2 (Passage n, test) |
| Background_2 |
Background signal intensity Ch2 (Passage n, test) |
| Adj.Intensity_2 |
Normalized signal intensity Ch2 (Passage n, test) |
| VALUE |
normalized signal intensity ratio passage n/passage 1 |
