Fig. 1

Schematic representation of the experimental approach. Series of cryostat sections of post-mortem brain tissue, dissected from subcortical NAWM of 18 MS patients and 9 control subjects, were used for RNA extraction. Sections preceding and following these series were stained by immunohistochemistry for proteolipid protein and HLA-DP, −DQ, −DR to confirm the absence of MS lesion pathology. In parallel, RNA was extracted from snap-frozen tissue dissected from a diversity of anatomical regions from control and MS brains, including MS lesions and NAWM, as well from tonsil, which was pooled and used to generate common reference cRNA. Common reference cRNA was co-hybridized to every microarray slide to allow for accurate comparison of expression levels across different cDNA microarray experiments. In this way, a ratio between the experimental and reference material could be calculated for every spot, and expression levels across different hybridizations could be compared. These data were subjected to WGCNA to identify clusters of co-regulated genes associated with HPA-axis activity and severity of MS. Furthermore, the data were used for group-wise comparisons between control subjects and MS patients subdivided into subgroups with high and low cortisol levels or subgroups with severe and mild disease using LIMMA