Labchip gx

A FFPE section stained with hematoxylin and eosin (H&E) was examined by a head and neck pathologist to confirm the presence of invasive tumor. For RNA purification (High Pure RNA Isolation Kit, Roche Diagnostic), either 2‐4 × 10 μm scrolls were obtained or if the tumor percentage was <70%, macro‐dissection was performed on multiple air‐dried sections. RNA quantity and integrity were determined with the NanoDrop ND‐2000 UV‐Vis Spectrophotometer (Thermo Fisher Scientific) and with either a LabChip GX (PerkinElmer) or a TapeStation (Agilent Technologies). A minimum of 200 ng of RNA from 44 OTSCC and six non‐cancer samples was used to measure the expression of 730 immune‐related genes and 40 housekeeping genes using the nCounter® platform (NanoString Technologies) and the PanCancer Immune Profiling Panel according to the manufacturer's instructions. PD‐1 expression is not assessed by this platform. Briefly, input RNA was hybridized to target sequence‐specific capture probes and fluorescent‐labeled reporter probes for 15‐19 hours at 65°C. The mRNA‐probe complexes were washed, immobilized, and quantified by the nCounter digital analyzer according to manufacturer's instructions.

Chromosome conformations identified using the DNA CHIP were confirmed using nested PCR performed as recently described (11 (link)). Briefly: “Sequence specific oligonucleotides were designed around the chosen sites for screening potential markers by nested PCR using Primer3. All PCR amplified samples were visualized by electrophoresis in the LabChip GX, using the LabChip DNA 1K Version2 kit (Perkin Elmer, Beaconsfield, UK) and internal DNA marker was loaded on the DNA chip according to the manufacturer’s protocol using fluorescent dyes. Fluorescence was detected by laser and electropherogram read-outs translated into a simulated band on gel picture using the instrument software. The threshold we set for a band to be deemed positive was 30 fluorescence units and above.” (11 (link)).

RNA isolation, sample preparation, and sequencing was conducted at the University Medical Center Groningen in Groningen, the Netherlands. Each sample was homogenized with zirconia/silica beads in the BeadBeater machine (BioSpec products, Inc.). After homogenization, total RNA was extracted and purified using an RNeasy microkit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. An initial quality check of the samples by capillary electrophoresis and RNA quantification for each sample was performed using the LabChip GX (PerkinElmer, Waltham, Massachusetts, USA). Samples with a minimum amount of 7 ng non-degraded RNA were selected for subsequent sequencing analysis. Sequence libraries were generated using the TruSeq RNA sample preparation kit from Illumina (San Diego, USA) using the Sciclone NGS Liquid Handler (Perkin Elmer). To remove contamination of adapter-duplexes, an extra purification of the libraries was performed with the automated agarose gel separation system Labchip XT (Perkin Elmer). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2000 using default parameters (single read 1x100bp) in pools of 10 or 11 samples. Processing of the raw data, including a demultiplexing step, was performed using Casava software (Illumina) with standard settings.