Sample Collection

Visit prior to colonoscopy: The volunteer brings in doctor’s office before colonoscopy (a) fecal sample on ice, (b) full blood test exams; and (c) the filled in QUESTIONAIRE; the doctor goes through to make sure the volunteers added all the correct information. Personal data will not be distributed to the researchers, but Prof. Apidianakis. Coded demographic data will be given to the researchers for analysis. The volunteer is given verbal and written advice on how to prepare for colonoscopy. Colonoscopy visit: (1) The doctor will collect a blood sample just before anesthesia for colonoscopy. Processing involves: (i) the separation of serum from WBCs, (ii) DNA extraction from WBCs, and (iii) storage of serum and DNA at -80 °C. (2) During colonoscopy the doctor collects 10 pinch biopsies (10-20mg each) of Normal-Appearing Mucosa (NAM; ≥15cm away from any tumor): (a) 2 from the rectum, (b) 2 from the sigmoid colon, (c) 2 from the descending colon, (d) 2 from the transverse colon, and (e) 2 from the ascending colon. Each time the endoscope moves from one section of the gut to another the tip will be washed by shaking for 10 sec. in: (i) ddH2O, (ii) 70% ethanol, (iii) ddH2O. (3) pinch biopsies are added in pre-weighed, pre-labeled (6-digit code and date) 2 ml RNase, DNase free tubes, containing 200μl 0.1mm & two 3mm zirconium beads. Tubes should be immediately embedded in liquid nitrogen at -80 ⁰C and then stored at -80 ⁰C. From processed samples, DNA will be stored at -20 °C and RNA and proteins at -80 °C. (4) In addition, the doctor will remove and collect (in formalin) any tumors to be assessed histologically by our collaborating histopatholostists. For total RNA, DNA and Protein extraction we will use the “RNA/DNA/Protein Purification Plus Kit” (Norgen Biotek) or the “NucleoSpin TriPrep” (Macherey-Nagel) per manufacturer’s instructions. Before starting we (a) wipe bench area with 75% ethanol, RNA away and 75% ethanol; (b) Precool homogenizer for 20 min in the cold room; (c) Add lysis buffer to frozen samples (d) Homogenize sample for 2 min at 5,000 rpm at 4 ⁰C. DNA is stored at -20 ⁰C, while RNA and proteins are stored at -80 ⁰C.