Anti-biotin magnetic beads pre-loaded with biotinylated antibodies against human CD4 and CD8 (Miltenyi Biotec) were used to isolated specific T cells from PBMCs. The all samples included in this study showed purity more than 90% by cytometry analysis. Peripheral blood mononuclear cells (PBMCs) were isolated from MS patients and healthy controls by centrifugation using Ficoll-Paque PLUS, and CD4+ and CD8+ T-cells were immunomagnetically purified using the positive cell Isolation Kit (Miltenyi Biotec). Total RNA was extracted using TRIZOL-LS (Invitrogen), treated with DNase and purified using the RNAeasy kit (Qiagen, Valencia, CA, USA). RNA quality was assessed by agarose gel electrophoresis and quantified using a NanoDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE, USA).
Label
Cy3
Label protocol
Cy3-labelled cRNA was generated using the “Quick Amp Labeling Kit, one-color” (Agilent, 5190-0442), following the manufacturer’s protocol. Briefly, 1µg total RNA from each studied sample was spiked with a control RNA (“One Color RNA Spike-In Kit”, Agilent, 5188-5282) and reverse transcribed using T7 oligo(dT) primers, followed by synthesis of the second complementary DNA (cDNA) strand and purification of double-strand cDNA with QIAquick columns (Qiagen). Cy3-labelled cRNA was then generated by an in vitro transcription reaction using T7 RNA polymerase and Cyanine 3-CTP and labeling efficiency was measured using a NanoDrop 1000 spectrophotometer.
Hybridization protocol
The cRNA was purified on an RNeasy column and 10µg were fragmented and hybridized to microarrays overnight (17h at 65oC and 4 RPM) in SureHyb chambers placed in a rotator oven incubator (Agilent, G2534A, G2530-60029 and G2545A), using the “Gene Expression Hybridization Kit” (Agilent, 5188-5242). After posthybridization washes (“Wash Buffers 1 and 2 Kit”, Agilent, 5188-5327), arrays were dried and analyzed.
Scan protocol
After processing, microarray slides were scanned at 535 nm and 5um/pixel resolution using a GenePix 4000B scanner and the software “GenePix Pro 6.0” (Molecular Devices, Sunnyvale, CA, USA).
Description
All samples were obtained by positive selection (using MACS Kits, from Miltenyi Biotec) to purities above 90% (% of CD4 or CD8+ cells, determined by flow cytometry).
Data processing
Images were analyzed with the software “Agilent Feature Extraction 8.5” and extracted expression values (gProcessedSignal) were further normalized to the 75 percentile expression value, considering all non-control features (ControlType=0). Following normalization, replicate probes were averaged, resulting in the final set of 41,000 unique probes.
Gene expression analysis of T cell subsets from MS patients undergoing autologous hematopoietic stem cell transplantation reveals significant changes in immune function
