DIE-RNA: a reproducible strategy for the Digestion of normal and injured pancreas, Isolation of pancreatic cells from genetically engineered mouse models and Extraction of high quality RNA
- 1Université catholique de Louvain, de Duve Institute, Belgium
- 2Flow Cytometry and Cell Sorting Facility (CYTF), de Duve Institute, Belgium
The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 µg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cells RNA. We thus validated the DIE (Digestion, Isolation and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.
Keywords: Flow Cytometry, Inflammmation, Pancreas, RNA quality control, cell isolation, Pancreatitis
Received: 13 Nov 2017;
Accepted: 07 Feb 2018.
Edited by:Peter Hegyi, University of Szeged, Hungary
Reviewed by:Savio George Barreto, Medanta The Medicity, India
Jens T. Siveke, Division of Translational Oncology (Solid Tumors), German Cancer Consortium (DKTK) site Essen, Germany
Matthias Sendler, Universitätsmedizin Greifswald, Germany
Copyright: © 2018 Assi, Dauguet and Jacquemin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Dr. Mohamad Assi, de Duve Institute, Université catholique de Louvain, Brussels, Belgium, email@example.com
Prof. Patrick Jacquemin, de Duve Institute, Université catholique de Louvain, Brussels, Belgium, firstname.lastname@example.org