Title: Affymetrix CEL analysis. Description:
This document describes Affymetrix recommended labeling protocols for use with GeneChip Exon Arrays.
Biotinylated cRNA (15 ug) was hybridized to 12 Affymetrix GeneChip Mouse Genome MOE430 2.0. using the one-cycle target-labeling kit according to the manufacturer's instructions.
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 15, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 24)
Dendritic cells were derived from female adult mice aged 8-12 weeks. The cells were cultured in a medium containing Iscove's Modified Dulbecco's Media (IMDM),10% fetal calf serum, glutammin, penicillin, streptomycin, and recombinant GM-CSF (10 ng/ml). Maintained for 4 hours at 37 degrees C with 5% CO2.
(Parameters: start time = 16, time unit = hours, min temperature = 45, temperature unit = C)
Cells (1 x 106 cells/ml) were stimulated in complete medium (IMDM with 10% FBS) with MSU crystals (monosodium urate, 250 ug/ml, Alexis) for 4 hours. MSU preparations were assayed using the limulus amebocyte lysate (LAL) test and were endotoxin-free.
Total RNA was isolated by double extraction using Trizol® (Invitrogen, Carlsbad, CA, USA), followed by the RNeasy clean-up procedure (Qiagen, Valencia, CA, USA). RNA purity and integrity were assessed by spectrophotometer and Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. Only high-quality RNA samples with a 260/280 ratio <1.9 and RIN >8.5 were used for microarray analysis.
(Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)