5 µm FFPE tissue sections of normal colorectal mucosa or carcinoma samples from Lynch syndrome individuals were deparaffinized and microdissected manually. Tumor areas with at least 80% tumor cell content were selected. Then, samples were digested with Proteinase K overnight. Nucleic acids were extracted with the automated system Promega Maxwell 16 LEV RNA FFPE Purification Kit (Promega, Madison, WI), treated with DNaseI at room temperature for 15 min (TURBO DNA-free ™ Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and used for RNA-based library preparation. The RNA concentration was measured fluorimetrically using the QuBit 2.0 RNA high sensitivity kit (ThermoFisher Scientific, Waltham, MA) with a minimum of 1 ng/ml set as quality threshold.
Label
biotin
Label protocol
standard protocol
Hybridization protocol
Probes and target transcripts were hybridized at 65°C for 18 hours according to the manufacturer's recommendations.
Scan protocol
The samples were scanned at maximum resolution on the nCounter Digital Analyzer.
Data processing
Sample normalization of the gene expression data was performed by fitting a linear model to negative and positive controls and subsequent housekeeping gene normalization. For the latter, the 20 panel genes with the lowest coefficient of variation and an expression level of at least 100 in the TCGA lung ADC dataset were used as housekeepers (AKT1, API5, DNAJC14, EIF2B4, ELA, ERCC3, GLUD1, HDAC3, HMGB1, IFNAR1, MLH1, OAZ1, PUM1, RIPK1, SF3A1, STAT3, TBC1D10B, TLK2, TMUB2, and UBB).
