genetic background: C57BL/6 gender: male cell type: primary murine fibroblast-like synoviocytes (FLS) genotype: human TNF transgenic and knockout in Rsk2 gene (Rsk2-/y FLS) flow cytometric cell sorting: CD90.2pos/VCAM1pos/CD11bneg
Biomaterial provider
Own in mouse breeding at the Nikolaus-Fiebiger-Centre, Erlangen.
Treatment protocol
Murine fibroblast-like synoviocytes (FLS) were isolated from the hind paws of hTNFtg and hTNFtg;Rsk2-/y (background: C57BL/6) 6 - 9 week-old mice via collagenase digestion. Cells were cultured in DMEM medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and gentamycin (0.5 µg/ml). All cell cultures were carried out at 37 °C in 5% CO2. The cells of two mice were pooled and cultivated for at least 3 passages before usage. For characterization, the cells were tested for the surface expression of CD90.2 and VCAM1 by flow cytometric analysis (> 95%), whereas CD11b as a monocytic marker had to be absent (< 5%).
Growth protocol
Murine fibroblast-like synoviocytes (FLS) were isolated from the hind paws of hTNFtg and hTNFtg;Rsk2-/y (background: C57BL/6) 6 - 9 week-old mice via collagenase digestion. Cells were cultured in DMEM medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and gentamycin (0.5 µg/ml). All cell cultures were carried out at 37 °C in 5% CO2. The cells of two mice were pooled and cultivated for at least 3 passages before usage. For characterization, the cells were tested for the surface expression of CD90.2 and VCAM1 by flow cytometric analysis (> 95%), whereas CD11b as a monocytic marker had to be absent (< 5%).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).
Label
Biotin
Label protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C, rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.
Hybridization protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C, rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.
Scan protocol
Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software version 1.4 from Affymetrix.
Description
Gene-expression profiling of primary murine hTNFtg FLS, Rsk2-/y; chip 1. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)
Data processing
Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 chips of group 1 were compared to the 3 chips of group 2 and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 9 SLR values of Experiment group vs. Baseline group and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls of Experiment group vs. Baseline group have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria. These are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (in one direction only).
