BioProject

To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. More...

To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. We studied 4 monozygotic twin pairs discordant for disease, with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip® Overall design: For gene expression profiling, peripheral blood mononuclear cell (PBMC) were obtained from leukopheresis by density centrifugation over Ficoll-hypaque according to standard procedures. Briefly, heparinized blood was diluted with 1 volume of RPMI medium and gently layered over the Fycoll. Following centrifugation at 660 g for 30 minutes, cells at the interface of the gradient was collected and washed 3 times. Highly purified CD4+ and CD8+ (purity >95%) cell subsets were then sorted by flow cytometry on MoFlo high speed cell sorter (Beckman Coulter, Fullerton, CA) and frozen in Trizol (Invitrogen). Total RNA was extracted using TRIzol® (Invitrogen), according to the manufacturer's instructions. RNA quality and purity were assessed with the use of the RNA 6000 Nano assay on Agilent 2100 Bioanalyzer (Agilent). Total RNA from each sample was prepared using GeneChip® one Cycle cDNA Synthesis kit and GeneChip® IVT Labeling kit (Affymetrix), according to the manufacturer’s protocols. Briefly, starting from 2.5µg total RNA, biotinylated cRNA was produced by one cycle of reverse transcription and in vitro transcription (IVT). Biotinylated cRNA was fragmented and hybridized for 16 h at 45°C onto GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix). The statistical analysis was performed couple by couple using the Rosetta Resolver® system (Rosetta Biosoftware). The fold-change of each gene was calculated by comparing gene expression in the affected twin with that in the unaffected twin and the genes with the same trend in at least 3 twin pairs out of the 4 studied were selected as candidates to be validated by real-time RT-PCR. Less...