Rwe isolated total RNA using the Trizol Reagent (Invitrogen) then purified using the RNeasy Mini Kit (Qiagen). RNA from spleen cDCs, PDCs and IKDCs were processed using the two-round RNA amplification protocol described by Affymetrix (Affymetrix GeneChip Expression Manual Small Sample_2) (http://www.affymetrix.com/support/technical/technotes/smallv2_technote.pdf).
Label
biotinylation then SA-PE as readout
Label protocol
We label anti-sense cRNA using BioArray RNA High Yield Transcript Labelling kit (ENZO Life Sciences Inc).
Hybridization protocol
hybridized 10 μg of total fragmented cRNA to the Affymetrix murine genome GeneChip array U74 set for 16 h at 45 oC with constant rotation (60 rpm).
Scan protocol
Fluorescence was detected using the Affymetrix- GS3000 GeneArray Scanner
Description
image analysis of each GeneChip was done through the GeneChip Operating System software from Affymetrix (GCOS1.1.1), using the standard default settings. We used global scaling for comparisons between different chips, scaling all probe sets to a user defined target intensity (TGT) of 150. To ascertain the quality control of the total RNA from each sample, we used the Agilent Bioanalyzer Lab on a Chip technology, and confirmed the rRNA ratios and clean run patterns of each sample. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess QC of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: scaling factor values within comparable range; low background values (between 20 and 100); high percentage of present calls (between 25 and 50); consistent 3’/5’ ratios of Gapdh as representation of housekeeping genes, and presence or absence of Bio B and C as internal spike controls.
