Matured DC (mDC) were transduced with the recombinant Adenovirus vaccine (encoding three full length antigens: Tyrosinase, MART-1, MAGE-A6) for 3hrs at 37°C.
Growth protocol
Patient immature dendritic cells were generated from elutriated monocytes using GM-CSF and IL-4 for 5 days. On day 6, immature DC were matured using IFNG and LPS for 24hrs. 24hrs. Post-maturation, mDC were transduced with the adenovirus vaccine for 3hrs.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by using RNeasy kit (Qiagen, Germantown, , MD, USA). The RNA concentration was measured using Nano Drop ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA) and RNA quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 300ng of total RNA per sample was amplified according to manufacturer's instructions (Ambion WT Expression Kit).
Label
biotin
Label protocol
cDNAs were fragmented, biotinylated, and hybridized to the GeneChip Human Gene 2.0 ST Arrays (Affymetrix WT Terminal Labeling Kit).
Hybridization protocol
the arrays were washed and stained on a GeneChip Fluidics Station 450
Scan protocol
Scanning was carried out with the GeneChip Scanner 3000 and image analysis with the Affymetrix GeneChip Command Console Scan Control(all from Affymetrix, Santa Clara, CA).
Description
Gene expression data from mDC, melanoma
Data processing
R/Bioconductor was used for the primary analysis. (Oligo & Limma Packages)
