The Spi-B/POU2AF1 gene expression analysis in peripheral blood mononuclear cells (PBMCs) could be performed at baseline and after 24 months of therapy in 13 patients with RRMS and 10 age- and sex-matched HCs. PBMCs were isolated from peripheral blood by density gradient centrifugation (Lympholyte Separation Medium, Cedarlane, Hornby, Ontario, CA, USA) and stored at −80°C until use.
Total RNA was extracted from PBMCs (RNAeasy Mini extraction kit, Qiagen), quantified and reverse-transcribed to cDNA, as previously described (19 (link)). RNA quality and purity were assessed by the analysis of spectrophotometric of 260/280 (≥2.0) and 260/230 ratio (range of 2.0–2.2). Pre-designed and validated assays (Thermo Fisher Scientific) were used for relative quantification of the target genes (Spi-B and POU2AF1) and for two previously selected housekeeping genes (GAPDH, YWHAZ). Quantitative PCR (qPCR) experiments were performed on CFX Touch real-time PCR (Bio-Rad) in triplicate, including the positive and non-template controls. All procedures were carried out under stringent conditions to avoid contamination. A relative expression analysis was performed by qBase+ software [Version: 3.0, Biogazelle], using a comparative cycle threshold method (20 (link)). The change in gene expression fold compared with baseline (delta fold) was calculated for each subject.
Total RNA was extracted from PBMCs (RNAeasy Mini extraction kit, Qiagen), quantified and reverse-transcribed to cDNA, as previously described (19 (link)). RNA quality and purity were assessed by the analysis of spectrophotometric of 260/280 (≥2.0) and 260/230 ratio (range of 2.0–2.2). Pre-designed and validated assays (Thermo Fisher Scientific) were used for relative quantification of the target genes (Spi-B and POU2AF1) and for two previously selected housekeeping genes (GAPDH, YWHAZ). Quantitative PCR (qPCR) experiments were performed on CFX Touch real-time PCR (Bio-Rad) in triplicate, including the positive and non-template controls. All procedures were carried out under stringent conditions to avoid contamination. A relative expression analysis was performed by qBase+ software [Version: 3.0, Biogazelle], using a comparative cycle threshold method (20 (link)). The change in gene expression fold compared with baseline (delta fold) was calculated for each subject.
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