Title: Affymetrix CEL analysis. Description:
RNA was isolated using the RNeasy Plant Mini Protocol (QIAGEN, Clifton Hill, Australia) and the quality of the RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) where spectrophotometric analysis was carried out to determine the A260 to A280 ratio and the concentration. For qRT-PCR, cDNA synthesis was carried out using the Biorad (Perth) iScript cDNA synthesis kit according to manufacturers instructions. For arrays, the cRNA was prepared from 3 ug of total RNA almost exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Title: GeneChip hybridization. Description:
nucleic acid extraction protocol
Standard Affymetrix protocol for tota RNA isolation
Cells from spleen were prepared for flow cytometry as previously described (Chatzidakis, I et al., 2007. An essential role for tnf in modulating thresholds for survival, activation, and tolerance of cd8+ t cells. J Immunol 178:6735-45)