age at diagnosis: 74 Sex: male histology: adenocaricnoma smoking status: former-roker treatment after surgery of bm: Radiotherapy
Extracted molecule
total RNA
Extraction protocol
Tissue sections of 5 μm were stained with hematoxylin and eosin (H&E), and examined by a pathologist. Total RNA was extracted from 10-12 sections of 10 μm thickness. RNA was stored in RNase/DNase-free water at -80°C. The nCounter® PanCancer IO 360™ Panel was used for NanoString targeted gene expression analysis. The total RNA was isolated from tumor tissue using the RNeasy® FFPE isolation kit (Qiagen, Hilden, Germany). The RNA quantity and quality were measured using the Agilent 2100 BioAnalyzer (Santa Clara, CA, USA), RNA degradation was calculated using percentages of fragments of 300-4000 nucleotides. The 300 ng of good quality RNA, with a maximum of 7 μL was used for hybridization with the panel probes for 17 hours at 65°C using a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA).
Label
n.a.
Label protocol
n.a.
Hybridization protocol
For each sample, 300 ng of good quality RNA, with a maximum of 7 μL was used for hybridization with the PanCancer IO 360™ probes for 17 hours at 67⁰C, following the manufacturing procedure (NanoString Technologies Inc., Seattle, WA, USA).
Scan protocol
The nCounter FLEX platform was used and genes were counted by scanning 490 Fields-of-view (FOV).
Data processing
Data analysis was performed using the advanced analysis module (version 2.0) of nSolver software (version 4.0, NanoString Technology).
