EAE immunization: C57BL6/J mice were immunized with the Hooke Kit™ MOG35-55/CFA emulsion PTX (EK-2110, Hooke Labs, St Lawrence, MA), which contains 200 µg myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified in 200 µl Complete Freund’s Adjuvant (CFA). The emulsion was injected subcutaneously at two sites followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) in phosphate buffered saline (PBS), the first 1-2 h after MOG35-55, and the second 24 h after MOG35-55. Sham control mice received a subcutaneous injection of CFA without MOG35-55. Mice were treated with R-flurbiprofen (10 mg/kg/d) or vehicle. Treatments were continuously administered via the drinking water starting 5 days after immunization. Drinking bottles were exchanged daily with freshly prepared drug or vehicle. Therapy was monitored by weighing water bottles and by analysis of plasma concentrations of R-flurbiprofen.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from homogenized tissue with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit according to the protocol provided in the RNeasy tissue Mini Kit. The quality of total RNA was checked by gel analysis using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples with RNA index values greater than 8.5 were selected for expression profiling. RNA concentrations were determined using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE)
Label
Biotin
Label protocol
The laboratory work was done in the Genomics and Proteomics Core Facility at the German Cancer Research Center, Heidelberg, Germany (DKFZ). Biotin-labeled cRNA samples for hybridization on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., 1992). In brief, 100 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the Illumina® Total Prep™ RNA Amplification Kit (Life Technologies). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. (Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, Zettel M, Coleman P. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. U. S. A. (1992) 89:3010–3014).
Hybridization protocol
Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned.
Scan protocol
Microarray scanning was done using an iScan array scanner. Data extraction was done for all beads individually, and outliers were removed when > 2.5 MAD (median absolute deviation). All remaining data points were used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated.
Description
EAE-experimental autoimmune encephalomyelitis; F-R-Flurbiprofen; Veh-Vehicle (i.e. tap water); MS-Multiple sclerosis model; KONT-negative control; s.c.-subcutaneous; p.o.- perorally; MOG 35-55-peptide of myelin oligodendrocyte glycoprotein used for immunization; CFA-complete Freund adjuvant
Data processing
Microarray scanning was done using an iScan array scanner. Data extraction was done for all beads individually, and outliers are removed when the absolute difference to the median is greater than 2.5 times MAD(2.5 Hampel’s method). All remaining bead level data points are than quantile normalized [1]. As test for significance the student’s t-test was used on the bead expression values of the groups of interest. In the case of significance of expression against background we tested for greater than all negative beads for this sample and in the case of comparing separate groups we tested for inequality of the means of the groups. In both cases Benjamini-Hochberg correction [2] was applied to the complete set of p-values of all ProbeIDs on the chip. The average expression value was calculated as mean of the measured expressions of beads together with the standard deviation of the beads. [1] Probe Level Quantile Normalization of High Density Oligonucleotide Array Data, Ben Bolstad, Division of Biostatistics, University of California, Berkeley December 2001 [2] Y. Benjamini and Y. Hochberg (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society B, Vol. 57, 289–300.
