For each patient, the NSABP Department of Pathology produced serial 10 μM sections from selected formalin-fixed paraffin-embedded (FFPE) tumor samples and sent anonymized, matched unstained slides as well as hematoxylin and eosin (H&E) stained slides to the Genomics and Epigenomics Shared Resource (GESR) at Georgetown University Medical Center (GUMC). At the GUMC Histopathology and Tissue Shared Resource (HTSR), the samples underwent pathological examination to confirm diagnosis and identify malignant tissue. Using the matching H&E slides as templates, tumor-containing areas were macrodissected from the unstained slides and processed for RNA isolation.
After deparaffinization, the macrodissected tissues were processed using the Roche High Pure FFPET RNA Isolation Kit (Roche Molecular Systems, Pleasanton, CA). The concentration of the extracted RNA was estimated by ultraviolet-visible spectrophotometry (NanoDrop 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA) to ensure sample purity (optical density 260/280 nm ratio 1.7–2.5). We assessed RNA quality using the Agilent RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the degree of RNA integrity with the Agilent 2100 Expert Software, as previously described (12 (link)).
After deparaffinization, the macrodissected tissues were processed using the Roche High Pure FFPET RNA Isolation Kit (Roche Molecular Systems, Pleasanton, CA). The concentration of the extracted RNA was estimated by ultraviolet-visible spectrophotometry (NanoDrop 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA) to ensure sample purity (optical density 260/280 nm ratio 1.7–2.5). We assessed RNA quality using the Agilent RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the degree of RNA integrity with the Agilent 2100 Expert Software, as previously described (12 (link)).
