Tissue Preparation and DNA Extraction

We utilized a total of 10 CRC RPO + cases for molecular study. Tumor specimens were harvested following surgical resection and snap frozen in liquid nitrogen. Up to three 1-cm² full-thickness specimens from the center and edge of the cancer were collected. In addition to cancer tissues, three 1-cm² normal colonic epithelium full-thickness specimens at least 8 cm from the polyp/tumor margin were harvested. Regions were identified by an expert gastrointestinal pathologist (T.J.S.) on a hematoxylin and eosin (H&E)–stained 4-μm–thick section of the frozen tissue blocks collected from surgical specimens from consented participants of the Biobank for Gastrointestinal Health Research (IRB 622-00, PI L.A. Boardman). Tumor tissue was macrodissected to enrich for tumor density (> 70%). All CRC RPO + tissues used here excluded subjects with a prior history of any malignancy, a family history of Lynch syndrome or familial adenomatous polyposis, and any other syndrome associated with hereditary CRC or inflammatory bowel disease. DNA was manually extracted utilizing PureGene chemistry (Gentra Systems, Minneapolis, MN). RNA was extracted using Qiagen MiRNeasy mini kit.