Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
nucleic acid labeling protocol
Genisphere Flashtag Biotin RNA labeling for Affymetrix microRNA assay
EAE was induced in female C57BL/6 mice (6–8 weeks old) via subcutaneous immunization with 100 ?L of 150 ?g myelin oligodendrocyte glycoprotein (MOG 35–55) peptide emulsified in complete Freund's adjuvant containing 4 mg·mL?1killed Mycobacterium tuberculosis. Following immunization, 200 ng of pertussis toxin was injected i.p. into mice on Day 0, followed by a 400 ng pertussis toxin i.p. injection on Day 2. For post-treatment studies, EAE was induced in naïve mice and 10 days later, these mice were completely randomized into groups that received treatment with vehicle or DIM (40 mg·kg?1). This treatment was continued every day (day 10 - day 25) throughout the remainder of the study.
nucleic acid extraction protocol
Affymetrix standard total RNA extraction
CD4 T cells were purified from mouse brains, before (naive) or on d 15 (EAE) of MOG35-55 peptide immunization and DIM-treated EAE mice , by selection with PE-conjugated mAb (all mAbs were purchased from Biolegend) using EasySep PE selection Kit (StemCell Technology) as previously described (Guan, H., et al. Journal of Immunology, 2009, 183: 172-180). Total RNA including microRNA was extracted from CD4+ T cells and PBLs using miRNeasy Mini kit (Qiagen) following the manufacturer's instruction.