Mirneasy ffpe tissue kit

Total RNA was extracted from tumor and corresponding normal tissue in paraffin-embedded blocks using the miRNeasy FFPE tissue kit (Qiagen) following the manufacturer's instructions. Total RNA was eluted in RNase-free water and stored at −20°C. The concentration and the quality of RNA were detected by spectrophotometer and by bioanalyser Agilent.

DNA and RNA were isolated from each sample, and their purities were measured; their quality was based on RIN, DIN, and DV₂₀₀. DNA was isolated using a QIAamp DNA formalin-fixed, paraffin-embedded (FFPE) Tissue Kit (Qiagen, Hilden, Germany). RNA was isolated using an miRNeasy FFPE Tissue Kit (Qiagen). The purity of the nucleic acid samples was assessed based on their spectrophotometric absorbance at 260–280 nm (A260/A280) using a NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RIN, DIN, and DV₂₀₀ indices and the concentrations of the extracted DNA and RNA samples were quantified using TapeStation 4150 (2015, Agilent Technologies, Santa Clara, CA, USA) based on the genomic DNA (gDNA) and RNA ScreenTape assays. Owing to instrument performance, RIN, DV₂₀₀, and DIN can provide quality measurements in samples with RNA concentrations >2.0 ng/μL and gDNA concentrations >3.0 ng/μL. Statistical analysis was performed on cases with evaluable RIN, DV₂₀₀, and DIN. Subsequently, to asses DNA quality and detect variants, NGS was conducted using an MiSeq sequencing platform. The AmpliSeq for Illumina Cancer HotSpot Panel v2 (50 target genes, 207 amplicons; Illumina Inc., San Diego, CA, USA) protocol was applied (Supplemental Information S5).

Per the manufacturer’s instructions, we extracted total RNA with the miRNeasy FFPE Tissue Kit (QIAGEN, Shanghai, China). In addition, we detected the purity and concentration of total RNA using NanoDrop 2000 (ThermoScientific, USA).