normalization data transformation
Array files were pre-processed, removing probes flagged as unreliable by Agilent Feature Extraction software and summarizing multiple probes for the same gene to a single value. Background subtraction was performed according to the half method, followed by quantile normalization. The procedure was carried out with the Agi4x44PreProcess package, part of the Bioconductor software suite (http://www.bioconductor.org) using the R programming language (http://www.r-project.org).
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies).
PBMC from healthy volunteers were obtained following approval by the local ethics committee and informed consent. DC were generated from adherent, monocyte-enriched PBMC fractions by first exposing adherent, monocyte-enriched PBMC fractions to optimized concentrations of GM-CSF + IL-4 for 6 days to generate imDC, followed by addition of a maturation cocktail consisting of IL-1I2, IL-6, TNF-alpha and PGE2 to harvest maDC on day 7.
nucleic acid hybridization
The Cy3- labeled cDNAs were hybridized overnight (17 hours, 65 C) to an Agilent Whole Human Genome Oligo Microarrays 4 x 44K using Agilent's recommended hybridization chamber and oven.
DCs were generated under GMP conditions from an initial apheresis of patients as follows: PBMCs isolated on Lymphoprep TM (Nycomed Pharma) were plated on NUNC Cell factories TM (Nalgene NUNC International) in RPMI 1640 (Cambrex, Belgium) supplemented with 2mM glutamine (Cambrex, Belgium), 20 µg/ml gentamycin (Refobacin „¢, Merck, Germany) and 1% heat inactivated autologous plasma. Adherent cells were cultured in 800 U/ml GM-CSF (available as a drug; Leukomaxa, Novartis, Germany) and 500 U/ml IL-4 (GMP quality, obtained from Strathmann or Cellgenix in Freiburg, Germany) for 6 days, then matured by adding 10 ng/ml TNF-alpha (available as drug; Beromuna, Boehringer Ingelheim, Germany) 2ng/ml IL-1beta (GMP quality of ACM-Biotech or Cellgenix), 100U/ml IL-6 (GMP quality from Novartis or Cellgenix, Germany) and 1 µg/ml prostaglandin E2 (available as drug: Minprostin E2, Pharmacia Upjohn, or Prostin E2, Pfizer, Germany).
250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec's undisclosed protocol.
RNA was extracted using SuperAmp Lysis Buffer (Milteny Biotec) and purified using a SuperAmp Preparation Kit, according to the manufacturer's instructions.