DNA microarray design and preparation
For the manufacturing of the arrays, 3'-aminomodified oligonucleotides were purchased from Metabion (Martinsried, Germany). Oligonucleotides were used at a final concentration of 10 μM in Spotting Buffer 1 (CLONDIAG chip technologies GmbH, Jena, Germany) and spotted using a Microgrid II spotting machine (Genomic Solutions Ltd., UK) following the procedure supplied by the manufacturer. Every probe was spotted redundantly two times on the array. After production, arrays were inserted into ArrayTube™ reaction vials.
Probe sequences were derived from published sequences using the Array Design software package by Clondiag Chip Technologies (Jena, Germany). In the additional files 1,2,3 tables show target genes, sources of the sequence data, as well as probe sequences and array layout. Consensus regions in the alignments of all available sequences of each target were chosen for the probe design. The resulting sequences were selected to be specific for the target and to have similar length, GC content and melting temperatures in order to yield comparable signal intensities. The final probe sequences were again blasted against the database  to exclude false-positive reactions due to possible cross-reactivities or false-negative reactions due to sequence variations.
The site directed oligonucleotide set for the linear amplification procedure consisted of 94 oligonucleotides. It was designed according to the initial alignments for the probe design as described above. For each target, a consensus region was identified which was situated 5 to 50 bp upstream of the probe binding site (see additional files). Sequences with similar physicochemical parameters were chosen from these regions and used for primer design. The final primer sequences were blasted against the database  avoiding possible cross-reactivities as well as sequence variations. Primer sequences are also listed in additional files (additional file 4). Oligonucleotides were purchased from Invitrogen (UK). They were used as a stock solution mixture with concentrations of 1 μM for every individual primer.
The development of the Affymetrix microarray was performed in an independent routine lab according to the manufacturers recommendations. 5 μg purified RNA was used as starting material for this purpose.
Total RNA from cells and tissues was isolated with TRIZOL® Reagent (Invitrogen, UK) according to the suppliers' instruction. RNA was re-dissolved in RNase-free water. The quality of the isolated RNA was controlled using non denaturing 1.25% agarose gel electrophoresis and the determination of the A260/A280 ratio (GeneQuant II, Pharmacia Biotech). Only samples exhibiting no RNA degradation and showing an A260/A280 ratio equal or above 1.8 were used for further applications. Preparations were stored at -80°C.
Labelling by reverse transcription of total RNA
For direct labelling of cDNA via reverse transcription 10 μg of total RNA was mixed with 1.5 μl oligo-dT15-primer (0.5 μg/μl, Promega, Mannheim, Germany) and sterile water (final volume 14, 6 μl), denatured for 15 minutes at 65°C, then chilled on ice for 5 minutes. After a short centrifugation step a mastermix consisting of 6 μl 5× First Strand Buffer (Invitrogen), 2 μl 0.1 M DTT (Invitrogen), 0.7 μl RNaseOUT (Invitrogen, 40 U/μl), 2.5 μl 5 mM dNTPs (dATP, dCTP, dGTP, Fermentas), 1 μl 3.25 mM dTTP, 1.4 μl 1 mM Biotin-16-dUTP (ROCHE, Penzberg, Germany) and 1.8 μl MMLV-RT (200 U/μl, Invitrogen) was added to get a final volume of 30 μl. The reaction mixture was incubated for 2 h at 37°C, and afterwards stopped by adding of 1 μl 200 mM EDTA (final concentration ca. 7 mM). The sample was precipitated with 0.1 vol 4 M LiCl/2.5–3 vol ice cold absolute ethanol, washed with ice cold 70% ethanol and resuspended in 10 mM EDTA.
Labelling with Klenow fragment
After First Strand cDNA synthesis with oligo-dT15-primer (Promega) and MMLV-RT (Invitrogen) the resulting cDNA was precipitated with LiCl/ice cold absolute ethanol, washed with ice cold 70% ethanol and dried. The pellet was resuspended in 68 μl deionisized water. This mixture served as template for a labelling reaction with Klenow-Fragment, exo-. We used the BioLabel DNA-Labelling Kit (Fermentas) and followed the protocol provided by the manufacturer to generate a biotin labeled hybridization sample. This DNA was purified by precipitation (LiCl/Ethanol) for further applications.
The First Strand cDNA was prepared with an oligo-dT T7-primer (5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC TTT TTT TTT TTT TTT TTT TTT TTT-3') and Superscript II RT (200 U/μl, Invitrogen) in a thermal cycler (10 minutes at 20°C, 60°C minutes at 37°C, final volume 10 μl). To generate the second strand an ice cold mastermix of 52.75 μl water, 7.5 μl 10×Second Strand buffer, 1.5 μl 10 mM dNTPs, 1 μl E. coli DNA-Ligase (5 U/μl, Fermentas), 2 μl DNA polymerase I (10 U/μl, Fermentas) and 0.25 μl RNase H (4.5 U/μl, Fermentas) was added to the cooled first strand tubes (final volume 75 μl). The double stranded cDNA (ds-cDNA) was purified by extraction with one volume phenol (once) and one volume of a chloroform/isoamyl alcohol mixture (24:1, once) and precipitated with 8 M NH4-acetate/ethanol at room temperature, washed twice with ice cold 70% ethanol and dried. The pellet was resuspended in 4 μl water. For In Vitro Transcription we used the Ambion Megascript kit (Ambion, USA). All components except the enzyme were allowed to come to room temperature. 1 μl of each component was added to the 4 μl ds-cDNA preparation and incubated for 16 h at 37°C. The resulting antisense RNA was purified with the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
Single primer amplification (SPA) and labelling with Klenow fragment exo- was performed according to the original protocol published in .
Labelling in the course of linear amplification reactions
Double stranded cDNA generated according to the Eberwine protocol described above served as template for labelling during the linear amplification reactions. For overall amplification we used a primer with the sequence of the T7-promoter (called SPA primer, analogue to the SPA-protocol).
For specific amplification of selected targets we used amounts of ds-cDNA-preparations representing original amounts of 50–100 ng total RNA. An ice cold mixture of water, 1.5 μl MgCl2 (50 mM), 5 μl 10×PCR buffer (Invitrogen), 1.5 μl 5 mM dNTPs (dATP, dCTP, dGTP), 0.5 μl 3.25 mM dTTP, 0.86 μl 1 mM Biotin-16-dUTP (ROCHE), 0.6 μl Taq polymerase (5 U/μl, Invitrogen) and 3 μl primer mix (1 μM each) resulting in a final primer concentration of 60 nM each was added to the template (final volume 50 μl). The amplification was performed in a thermal cycler according to the following protocol: 3 min 94°C, 40 cycles: 1 min 94°C, 1 min 62°C, 2 min 72°C, cooling to 4°C. The reaction mixture was divided to get material for duplicates and used for hybridization without further purification steps.
Hybridization and detection
The array tube was conditioned by washing three times with 500 μl distilled water and once with 500 μl hybridization buffer 1 (Clondiag, Jena) at 25°C for 5 minutes. All steps were carried out using a horizontal shaker with temperature regulation (550 rpm, Thermomixer compact, Eppendorf, Germany). Biotinylated spike controls (0.05 μl each, equivalent to 6.7 × 10-6-1 × 10-5 nmol) were added to the samples as external control for hybridization, conjugation of enzyme and signal development due to TMB precipitation.
The samples were denatured at 95°C for 10 min and briefly centrifugated at 13000 rpm to collect the sample. 200 μl of pre-warmed hybridization buffer (50°C) was added to each sample and the resulting mixture transferred into the array tube. Hybridization was allowed to proceed on the shaker for 3 h at 50°C and 550 rpm.
The array tube was washed with 500 μl 2×SSC/0.01% Triton (5 min, 30°C), 2×SSC (5 min, 20°C) and 0.2×SSC (5 min, 20°C). After blocking with 100 μl of a blocking solution (2% milk powder, freshly prepared in 6×SSPE/0.005% Triton, 15 min, 30°C) the array was incubated for 15 min at 30°C with 100 μl of a 1:10 000 dilution of HRP Streptavidin (1 mg/ml, Clondiag, Jena) freshly prepared in 6×SSPE/0.005% Triton. Following that the array was washed twice with 500 μl 2×SSC/0.01 Triton (2 min, 30°C), twice with 500 μl 2×SSC and once with 0.2×SSC (5 min, 20°C).
The peroxidase pecipitation reaction (100 μl peroxidase substrate, Clondiag, Jena) was monitored by the ATR01 array tube reader (Clondiag, Jena) at 25°C recording 60 images (one image per 10 sec). Data analysis was carried out using IconoClust software Version 2.2 (Clondiag) determining the signal intensity and the local background value of each spot. The local background absorbance was subtracted from the absorbance of the spots. Only the average values of redundant spot hybridization signals with amounts above 0.05 (mean - local background values) were considered as positive. Both spotted oligos of an examined gene had to be "positive" to be considered as expressed. If these conditions were met, the signal resulting from the hybridization with the oligo sequence situated closer at the 3'end of the RNA sequence was used for further calculations.
Real time PCR (QRT-PCR) experiments
QRT- PCR experiments were carried out using a MyiQ™ Single colour QRT- PCR detection system (BIORAD, Herculas, CA). Reaction mixtures contained: 1 μl cDNA, 9.5 μl water, 2 μl primer mix (sense and antisense, 10 μM each), and 12.5 μl iQ SYBR Green Supermix (BIORAD, final volume 25 μl). A dilution series (10-2 - 10-6ng) of the specific PCR product of interest was prepared to determine the standard curve (absolute quantification). Template free controls served as a test of primer quality (formation of dimers etc.). First of all the melting curve of each target was measured to determine the optimal temperature for real time analysis (e.g. ß-actin: 72°C, PAI-1: 83°C, matriptase: 89°C, TIMP3: 85°C). The samples were amplified according to the following protocol: 3 min 95°C, 35 cycles: 20 sec 95°C, 40 sec 58°C, 1 min 72°C, real time data registration for 8 sec at the specific temperature determined before for each target. All samples were measured in duplicates and the right formation of the products was verified by agarose gel electrophoresis (1% agarose, unknowns, standards and no template control, product sizes: matriptase 470 bp, PAI-1 687 bp, TIMP3 445 bp). Real time data analysis was carried out using the optical system software version 1.0 supplied with the MyiQ™ real time instrument (BIORAD).
Primary human fibroblasts were isolated from arthritic patients according to the rules of the ethic commission of the Friedrich Schiller University Jena. Cells were cultured in DMEM/high glucose, 10% FCS, gentamycine 0.5 ml/100 ml (in triplicate, 75 cm2 flasks) until 80% confluence was reached. The medium was removed; the cells were washed twice with FCS free medium and further cultured in FCS free medium overnight. The medium was then removed, and 4 ml of fresh FCS free medium was used for stimulation experiments. The first sample served as control (FCS free medium without any cytokine. The second sample was stimulated with 20 ng IL6/ml medium, the third sample was stimulated with 2 ng TGFß/ml medium. Stimulation was performed for 24 hours. Medium was removed, cells were harvested and RNA was isolated with TRIZOL® reagent as described above. RNA quality was checked by non denaturing agarose gel electrophoresis and used for preparation of double stranded cDNA according to the first steps of the Eberwine protocol (see above). Additionally, an established human fibroblast line was also used for expression analysis.