RNA extracts passing the following steps of quality control were considered as suitable for gene expression analysis: RNA concentration of at least 10 ng/µl, sufficient RNA purity with a A260/A280 in the range 1.7–2.3 and sufficient RNA integrity with at least 90% of the fragments longer than 100 nucleotides. Targeted mRNA expression profiling was performed using the NanoString nCounter gene expression platform (NanoString Technologies, Seattle, WA) using a 770-gene panel (PanCancer Human IO360 Panel) focused on the complex interplay between the tumor, the tumor microenvironment, and the immune response in cancer. Per sample, 100 ng of total RNA in a final volume of 5 μl were mixed with a 3′ biotinylated capture probe and a 5′ reporter probe tagged with a fluorescent barcode from the PanCancer IO360 gene expression code set. Probes and target transcripts were hybridized at 65°C for 18 hours according to the manufacturer’s recommendations. Hybridized samples were run on the NanoString nCounter preparation station using the high-sensitivity protocol, in which excess capture and reporter probes are removed and transcript-specific complexes are immobilized on a streptavidin-coated cartridge. The samples were scanned at maximum resolution on the nCounter Digital Analyzer.

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