Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.
Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.
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DOI：
10.1097/mpa.0000000000001717
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发表时间：
2021-01-01
期刊：
影响因子：
2.9
通讯作者：
Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer##
中科院分区：
文献类型：
--
作者：
Cruz-Monserrate Z;Gumpper K;Kaul S;Badi N;Terhorst S;Dubay K;Lesinski GB;Fisher W;McElhany A;Lara LF;Krishna S;Mace T;Higuita-Castro N;Ortega-Pineda L;Freitas MA;Hinton A;Yadav D;Hart PA;Pandol SJ;Ahmed S;Fatou B;Steen H;Conwell DL;Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer##
内窥镜胰腺功能测试用于诊断胰腺疾病，是发现更好地表征胰腺疾病的生物标志物的一个可行来源。然而，胰液(PF)含有降解生物分子的活性酶。因此，我们测试了保存方法和保存时间对蛋白质和核酸的完整性和质量的影响。我们从9名接受了内窥镜胰腺功能测试的受试者身上获得了PF。样品在采集时、冰上1、2、4小时后，或在4°C下储存一夜(含或不含核糖核酸酶或蛋白酶抑制剂)后进行快速冷冻。电泳法和质谱仪分析决定了蛋白质的丰度和质量，而核酸完整性决定了DNA和RNA的降解。冰上培养4小时后蛋白质降解增加，冰上培养2小时后DNA降解增加。添加蛋白水解酶抑制剂延缓了降解。与快速冷冻样品相比，RNA在所有条件下均显著降解。从PF来源的外体中分离的RNA显示出与从匹配的PF样本中分离的RNA类似的劣质。在收集PF并在收集后4小时内处理液体后立即添加蛋白酶抑制剂，可保持蛋白质和核酸的完整性，供下游分子分析使用。
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. 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. 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. 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.
影响因子：
2
作者：
Paulo, Joao A.;Lee, Linda S.;Wu, Bechien;Banks, Peter A.;Steen, Hanno;Conwell, Darwin L.
通讯作者：
Conwell, Darwin L.