After 8 days of culture, tDCs and mature DCs (mDCs) were generated by additional incubation with 10 ng/mL rmTNF-α (BD Biosciences, Mountain View, CA, USA) and 50 µg/mL type II collagen (Sigma-Aldrich) for 4 h, and with 1 µg/mL lipopolysaccharide (Sigma-Aldrich) and 50 µg/mL type II collagen for 24 h, respectively.
DCs were generated from bone marrow progenitors of 6-week-old DBA mice, as described previously[15, 20]. Briefly, bone marrow single-cell suspensions were prepared, the cells were washed, and the washed cells were cultured in RPMI 1640 supplemented with 10% FBS (Life Technologies, Grand Island, NY, USA), 20 ng/mL recombinant mouse GM-CSF (JW CreaGene, Gyeonggi, Korea), and 2 ng/mL rmIL-4 (JW CreaGene) at 37°C with 5% CO2. On Day 3, non-adherent cells were washed and re-fed using the same culture conditions. On Day 6, 50% of the culture medium was replaced with fresh medium containing the same concentrations of GM-CSF and IL-4.
Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, USA), purified using RNeasy columns (Qiagen, Valencia, USA) according to the manufacturers’ protocol. After processing with DNase digestion, clean-up procedures, RNA samples were quantified, aliquot and stored at -80°C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).
Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cRNA according to the manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP. After purification, the cRNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA).
750 ng of labeled cRNA samples were hybridized to each Mouse Ref-8 expression v.2 bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina, Inc., San Diego, USA). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual.
Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions.
The quality of hybridization and overall chip performance were monitored by visual inspection of both internal quality control checks and the raw scanned data. Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011.1 (Gene Expression Module v1.9.0)). Array probes transformed by logarithm and normalized by quantile method. Statistical significance of the expression data was determined using Fold change and LPE(Local Pooled Error) test in which the null hypothesis was that no difference exists among 2 groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm. For a DEG set, Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using DAVID (http://david.abcc.ncifcrf.gov/home.jsp)