Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids

Zobeida Cruz-Monserrate, Kristyn Gumpper, Sabrina Kaul, Niharika Badi, Samantha Terhorst, Kelly Dubay, Gregory B. Lesinski, William Fisher, Amy McElhany, Luis F. Lara, Somashekar Krishna, Thomas Mace, Natalia Higuita-Castro, Lilibeth Ortega-Pineda, Michael A. Freitas, Alice Hinton, Dhiraj Yadav, Phil A. Hart, Stephen J. Pandol, Saima AhmedBenoit Fatou, Hanno Steen, Darwin L. Conwell

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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 influence 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, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas 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 PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. Conclusions Adding PIs 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.

Original languageEnglish
Pages (from-to)17-28
Number of pages12
JournalPancreas
Volume50
Issue number1
DOIs
StatePublished - 2021

Bibliographical note

Funding Information:
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 influence 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, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass From the *Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, and †The James Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH; ‡Winship Cancer Institute, Department of Hematology and Medical Oncology, Emory University, Atlanta, GA; §The Elkins Pancreas Center, Michael E. DeBakey Department of Surgery, and ||Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX; ¶Department of Biomedical Engineering and #Department of Surgery, The Ohio State University, Columbus, OH; **Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, and ††Division of Biostatistics, College of Public Health, The Ohio State University, Columbus, OH; ‡‡Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh, Pittsburgh, PA; §§Digestive Disease Center, Cedars-Sinai Medical Center, Los Angeles, CA; and ||||Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA. Received for publication July 8, 2020; accepted November 3, 2020. Address correspondence to: Zobeida Cruz-Monserrate, PhD, Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, 2041 Wiseman Hall, 400 W 12th Ave Columbus, OH 43210 (e‐mail: zobeida.cruz-monserrate@osumc.edu). This study was supported by The National Pancreas Foundation (Z.C.-M.), the National Cancer Institute (NCI) R01CA223204 (Z.C.-M.), and the NCI and National Institute of Diabetes and Digestive and Kidney Diseases under award numbers U01DK108327 (D.L.C., Z.C.-M., P.H., L.L., S. Krishna, H.S.) and U01DK108306 (D.Y.). This study was also supported in part by the Pelotonia Fellowship Program (S. Kaul and K.D.), by grant P30 CA016058 NCI and by the ChiRhoClin Research Institute, Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Pancreas Foundation, the National Institutes of Health, the Pelotonia Fellowship Program, or the ChiRhoClin Research Institute. Z.C.-M., P.A.H., G.B.L., and D.Y. received pilot funds from ChiRhoClin Research Institute, Inc. The rest of the authors declare no conflict of interest. Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.pancreasjournal.com). Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/MPA.0000000000001717 spectrometry analysis determined protein abundance and quality, whereas 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 PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. Conclusions: Adding PIs immediately after collecting PFand processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.

Funding Information:
The authors thank The Ohio State University Genomics Shared Resource for the RNA and DNA sample quality control and the Proteomics Shared Resource funded by the NCI Cancer Center Support Grant P30 CA016058. We also thank the Division of Gastroenterology, Hepatology, and Nutrition clinical coordinators who assisted in subject recruitment and regulatory management of this study.

Publisher Copyright:
© Wolters Kluwer Health, Inc. All rights reserved.

Keywords

  • biomarkers
  • pancreatic cancer
  • pancreatic fluid
  • pancreatitis
  • sample degradation

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

Fingerprint

Dive into the research topics of 'Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids'. Together they form a unique fingerprint.

Cite this