RNA extraction and NanoString analyses were conducted as previously described (35). In short, H&E stained slides of HNSCC cases were examined to identify tumor regions and to estimate tumor cell content. FFPE material with a minimal tumor cell content of 20 % (median 65 %) was used for subsequent microdissection of RNA from 3 µm thick sections. RNA was then extracted according to manufacturer’s instructions on a Maxwell extraction system (Promega, Madison, WI) and quantified by Qubit (Thermo Fisher Scientific, Waltham, MA).
Label
NanoString
Label protocol
Labels are artificial numbers without semantical meaning
Hybridization protocol
For mRNA expression analyses 400 ng (concentration 100 ng/µl) isolated RNA per sample were hybridized on a nCounter Analysis System using a customized NanoString panel consisting of 231 genes according to manufacturer’s instructions (NanoString Technologies, Seattle, WA).
Scan protocol
Slides were scanned on a slide scanner (Aperio AT2, Leica Biosystems GmbH, Nussloch, Germany) and evaluated on a standard monitor (Fujitsu B24T-7, Fujitsu Limited, Tokyo, Japan, resolution 1920 x 1080) utilizing Aperio ImageScope x64 (version 12.4.0.7018; Leica Biosystems GmbH, Nussloch, Germany). One digitized high-power field (HPF) comprised 97464 µm2 which corresponded to a field diameter of 0.35 mm in light microscopy.
Dataset normalization was conducted with the ‘NanoStringNorm’ R-package, version 1.2.1 (36). The ‘stringR’ and ‘ggplot2’ packages in conjunction with the ‘pheatmap’ R-package (RRID:SCR_016418) were utilized for visualization and string manipulation (37,38). For differential expression analyses, the ‘LIMMA’ R-package was utilized (39,40). For computational analysis R (version 4.1.3) (41) and SPSS v25 (International Business Machines Corporation (IBM), Armonk, NY) were used.
