1. Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids
- Author
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Stephen J. Pandol, Amy L. McElhany, Darwin L. Conwell, Samantha Terhorst, Gregory B. Lesinski, Somashekar G. Krishna, William E. Fisher, Lilibeth Ortega-Pineda, Benoit Fatou, Saima Ahmed, Luis F. Lara, Kelly Dubay, Sabrina Kaul, Niharika Badi, Alice Hinton, Michael A. Freitas, Phil A. Hart, Hanno Steen, Dhiraj Yadav, Kristyn Gumpper, Natalia Higuita-Castro, Zobeida Cruz-Monserrate, and Thomas A. Mace
- Subjects
Time Factors ,RNase P ,RNA Stability ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Protein degradation ,Article ,Specimen Handling ,Workflow ,Secretin ,03 medical and health sciences ,chemistry.chemical_compound ,Ribonucleases ,0302 clinical medicine ,Endocrinology ,Pancreatic Juice ,Predictive Value of Tests ,Nucleic Acids ,Freezing ,Internal Medicine ,medicine ,Humans ,Protease Inhibitors ,Endoscopy, Digestive System ,Protease ,Hepatology ,Protein Stability ,Chemistry ,Pancreatic Diseases ,Proteins ,RNA ,Cold Temperature ,Pancreatic Function Tests ,medicine.anatomical_structure ,Biochemistry ,030220 oncology & carcinogenesis ,Proteolysis ,Nucleic acid ,030211 gastroenterology & hepatology ,Pancreas ,Biomarkers ,DNA ,DNA Damage - Abstract
OBJECTIVES: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influences the integrity and quality of proteins and nucleic acids. METHODS: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection, after 1, 2, 4 hours on ice, or after storage overnight at 4°C with or without RNase or protease inhibitors. Electrophoresis and mass spectrometry analysis determined protein abundance and quality while nucleic acid integrity values determined DNA and RNA degradation. RESULTS: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding protease inhibitors delayed degradation. RNA was significantly degraded under all conditions compared to the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. CONCLUSIONS: Adding protease inhibitors immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.
- Published
- 2021
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