4.5. Sample Processing: Pathology Review and Tumor Tissue
tumor, NCBs, necrotic, TCGA
TUMOR, PATHOLOGY, NECROTIC
RNAlater-submerged samples were stored at 4 °C for 72 h and then washed with RNAse-free phosphate-buffered saline for 6 min on dry ice. This last step was repeated three times. The biopsies were placed in the center of a 15 mm × 15 mm Tissue Tek disposable cryomold (Somagen, Edmonton, AB, Canada) to embed them in the OCT compound. OCT compound (Surgipath) was poured into the cryomold until the specimen was covered entirely. The cryomold was submerged for 30 s in a beaker containing 2-methylbutane (Fisher Scientific, Waltham, MA, USA) pre-cooled on dry ice, using tweezers to hold the cryomold to ensure that the sample remained horizontal. After the OCT solidified, the block was immediately stored at −80 °C. The preparation of tissue cryosections was performed using standard pathology laboratory procedures. Briefly, OCT blocks were placed for at least 30 min inside a cryostat at −20 °C prior to cutting to ensure the block’s temperature was optimal for cryosectioning. Sections 4–5 µm thick were cut onto a SuperFrost glass slide (Fisher) and stained with hematoxylin and eosin (H&E) in order to ensure the enrichment of genetic material from neoplastic cells in our samples. Histology quality control (HQC) and validation were performed on both paraffin and OCT embedded NCBs; H&E-stained sections were examined by a pathologist to determine the percentage of normal cells, tumor cells, and necrotic areas in each NCB. The threshold for acceptable tumor cell area for DNA/RNA extraction was set at >60% of the specimen, including <20% of necrotic cells, as suggested by the TCGA [