Total cellular RNA from polyclonal CD56+CD16+ in vitro activated NK subset (100U/ml IL-2 + 1ng/ml PHA)was pooled from 9 different donors was isolated and subjected to clean up protocol with RNeasy mini kits (Qiagen), according to the manufacturer’s specifications. The Quality of total RNA was assessed using an Agilent Bioanalyzer. First and second strand cDNA was prepared from 0.5µg RNA template, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was fragmented to uniform sizes (~100nt) CodeLinkTM Uniset Human 20K I Bioarrays (Amersham Biosceinces) were hybridized with each prepared cRNA target in duplicates and stained with Cy5-streptavidin and subsequently washed according to manufacturer’s instructions, and then scanned on an Axon GenePix 4000B scanner. Raw data files were obtained after analysis of scanned images with CodeLinkTM expression software (Amersham Biosciences).
