6 protocols
Title: Affymetrix CEL analysis. Description:
URL: http://www.affymetrix.com/support/technical/manual.affx
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 TrizolĀ® (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)