Samples stored at –80°C after overnight incubation in RNALater were used for the microarray hybridizations. Total RNA was extracted from the synovial biopsies using the Nucleospin® RNA II extraction kit (Macherey-Nagel), including DNase treatment of the samples. At least 1 µg total RNA could be extracted from 12 paired samples at T0 and T12 for further processing. RNA quality was assessed using an Agilent 2100 Bioanalyzer and RNA nanochips (Agilent technologies Inc).
Label
Biotin
Label protocol
Labeling of RNA (cRNA synthesis) was performed according to a standard Affymetrix® procedure (One-Cycle Target Labeling kit, Affymetrix UK Ltd., High Wycombe, UK); briefly total RNA was first reverse transcribed into single-stranded cDNA using a T7-Oligo(dT) Promoter Primer and Superscript II reverse transcriptase. Next, RNase H was added together with E.Coli DNA polymerase I and E. Coli DNA ligase, followed by a short incubation with T4 DNA polymerase in order to achieve synthesis of the second-strand cDNA. The purified double-stranded cDNA served as the template for the in vitro transcription reaction, which was carried overnight in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix. At the end of this procedure, the biotinylated complementary RNA (cRNA) was cleaned, and fragmented by a 35 -minute incubation at 95°C.
Hybridization protocol
GeneChip® Human genome U133 Plus 2.0 Arrays (spotted with 1,300,000 oligonucleotides, grouped in 54,675 probe sets informative for about 47,000 transcripts originated from not less than 39,000 genes, Affymetrix UK Ltd, High Wycombe, UK) were hybridized overnight at 45°C in monoplicates with 10 µg cRNA.
Scan protocol
The slides were then washed and stained using the EukGE-WS2v5 Fluidics protocol on the Genechip® Fluidics Station (Affymetrix) before being scanned on a Genechip® Scanner 3000.
Description
twenty patients with ra (17 women and 3 men, average age +/- sem: 52,6+/-3,8 years) were included in the study. All patients met the American College of Rheumatology classification criteria for the diagnosis of RA. All patients had active disease at the time of tissue sampling and were resistant to TNF blockade. They all had erosive changes imaged on conventional x-rays of the hands and/or feet. All of them had a swollen knee at inclusion. Rituximab therapy was administrated at a dose of 1,000 mg IV at baseline (T0) and at week 2, together with 125 mg IV Methylprednisolone. Clinical parameters at baseline (T0) and 12 weeks after the initiation of therapy (T12) was evaluated using DAS(28)-CRP scores and clinical responses were assessed using EULAR response criteria. Synovial biopsies were obtained by needle-arthroscopy of an affected knee from all patients at T0 and T12. For each procedure, 4 to 8 synovial samples were kept overnight at 4°C in a RNA stabilizing solution (RNALater, Ambion, Applied Biosystems, TX, USA) and then stored at –80°C for later RNA extraction. The same amount of tissue was snap-frozen in liquid nitrogen and kept at –80°C for immunostaining experiments on frozen sections. The remaining material was fixed in 10% formaldehyde and paraffin embedded for conventional optical evaluation and immunostaining of selected markers. All the experiments (RNA extraction, histology, immunohistochemistry) were performed on at least 4 biopsies harvested during every procedure in order to correct for variations related to the potential heterogeneous distribution of synovial inflammation. The study was approved by the ethics committee of the Université catholique de Louvain and informed consent was obtained from all patients.
Data processing
For the initial normalization and analysis steps, data were retrieved on Affymetrix GCOS software. The frequency of positive genes (genes with a flag present) was between 40 and 50% on each slide. After scaling of all probe sets to a value of 100, the range of the reported amplification scales was between 0,8 and 6,7. The signals yielded by the poly-A RNA, hybridization and housekeeping controls (GADPDH 3’/5’ ratio < 2.3 in all the slides) were indicative of the good quality of the amplification and hybridization procedures.
