Robert Szulawski1,2, Masato Nakazawa7, Kelly D. McCall*1,2, 3, 4, 5, 6, Calvin B.L. James*3,5, and Frank L. Schwartz*1,2, 6
1Department of Specialty Medicine, Ohio University Heritage College of Osteopathic Medicine, Athens, OH
2Diabetes Institute, Ohio University Heritage College of Osteopathic Medicine, Athens, OH
3Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Athens, OH
4Department of Biological Sciences, Ohio University College of Arts & Sciences, Athens, OH
5Molecular & Cellular Biology Program, Ohio University College of Arts & Sciences, Athens, OH
6Biomedical Engineering Program, Ohio University Russ College of Engineering & Technology, Athens, OH
7Office of Research and Grants, Ohio University Heritage College of Osteopathic Medicine, Athens, OH
*Des shared senior authorship.
BioTechniques, Vol. 60, No. 6, June 2016, pp. 293–298
Abstract
RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for usage in ex vivo expression studies. Tissue was extracted from female non-overweight diabetic (NOD) toll-love receptor 3 (TLR3)+/+ and (TLR3)-/- mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained along with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated along with the Leica LMD6000 laser capture microdissection (LCM) system to generate samples highly enriched in islet RNA. Actual time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a crucial role in the pathogenesis of type 1 diabetes mellitus (TIDM). This means represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small quantities of starting tissue devoid of the necessity for RNA pre-amplification. This has actually immediate implications for ongoing TIDM studies using the NOD mouse.
The female non-overweight diabetic (NOD) mouse is a widely studied and validated mammalian model of type 1 diabetes mellitus (TIDM). Our current knowing of the pathogenesis of TIDM in this animal model indicates that dendritic cells and macrophages, followed by T cells, are initial observable in and about pancreatic islets at ~4 weeks of age (1, 2). After sufficient T cell-mediated destruction of β-cell mass occurs, the onset of clinically apparent diabetes results, and 60%–100% of female NOD mice spontaneously create over t diabetes by 4–six months of age (3, 4), depending on environmental factors and variability between mouse colonies (5). There are substantial differences in the degree and nature of insulitis seen in the diabetic NOD mouse pancreas (diffuse and robust) compared along with the insulitis in a new-onset TIDM patient (often, focal and modest) (2, 6). Importantly, distribution of β cells in the islet differs between rodents and humans (7). Nevertheless, there are severe similarities between NOD and human TIDM, from the genetic linkage to individual human leukocyte antigen (HLA) subtypes and the supplement of severe histocompatibility complex (MHC) class 1 overexpression to TIDM aggressiveness (1), to the milieu of chemokines and immune cells surrounding or invading the islets (2, 8).
METHOD SUMMARY
Here we introduce a means for isolating pancreatic islets for usage in downstream expression studies on the pre-diabetic non-overweight diabetic (NOD) mouse. Flash-frozen samples were fixed and stained along with alcoholic cresyl violet solution and dehydrated in a manner that reduced RNase activity. RNA was isolated from stained islets by microdissection and used for multiplex real-time PCR.
We are particularly thinking about characterizing early (weaning to 10 weeks of age) molecular differences between wild-type NOD mice and toll-love receptor 3 (TLR3)–deficient mice (9, 10), as we have actually recently revealed that TLR3 is crucial for virus-mediated acceleration of TIDM (10). RNA extraction for gene expression studies from pancreatic islets of juvenile mice is complicated by the small size of the immature pancreas and a dearth of islets. The really higher RNase content of the exocrine pancreas additionally necessitates care in sample processing (11, 12). To our knowledge, laser capture microdissection (LCM protocols) for functioning along with rodent pancreatic tissue have actually not been specifically tailored to manage these problems (13, 14). Existing protocols extracting whole, intact islets from pancreatic sections appear to usage a lot more starting tissue compared to would certainly have actually been feasible in our experimental design or require pre-amplification to obtain an adequate quantity of input RNA for downstream studies. Commercially available RNA pre-amplification kits can easily sustain the true relative proportion of mRNA transcripts present in samples (15); however, this means of increasing RNA generate for transcriptome studies is expensive and adds additional steps to processing. Here we describe an improved protocol for isolating high-quality RNA from rodent pancreatic islets for multiple gene expression studies devoid of the necessity for RNA pre-amplification.
We tested this brand-new protocol by evaluating expression of CXC chemokine ligand 10 (CXCL10), because of the importance of this chemokine in the pathogenesis of TIDM. CXCL10 is inducible by IFN-gamma and serves as a chemoattractant for chemokine receptor CXCR3-positive cells (16). Up-regulation of CXCL10 and subsequent CXCR3-positive Th1 cell response is considered essential for the T-cell–mediated destruction of β cells that has actually classically been seen as the hallmark of TIDM (17–19). TLR3 activation has actually been revealed to mediate levels of CXCL10 expression in NIT-1 cultured β cells transfected along with a synthetic double-stranded RNA (dsRNA) (4), and TLR3 mRNA has actually been revealed to be up-regulated in autoimmune ailment in human and animal subjects (9, 20). Therefore, we wanted to investigate if there was a difference in expression of CXCL10 between wild-type (TLR3+/+) and TLR3-deficient (TLR3-/-) NOD mice in the pre-diabetic stage. We predicted that CXCL10 levels ought to be similar in the 2 sets of pet dogs that have actually not been exposed to virus because the insulitis scores of the 2 sets of pet dogs were the very same in our previous study (10), as was the fee of development of spontaneous diabetes (9, 10).
CXCL10 expression has actually been detected in the islets of NOD mice as early as 4 weeks of age, along with levels progressively increasing in the islets in the pre-diabetic period, followed by an eventual decrease after most β cells have actually been destroyed in the diabetic animal (3, 21). Some immunohistochemistry studies using pancreas biopsies isolated from human patients along with TIDM have actually revealed that there is little expression of CXCL10 in the exocrine pancreas at ailment onset however a marked raise of expression in the islets (19, 22, 23); yet another study has actually revealed involvement of the exocrine pancreas, along with close association of CXCL10 along with CD45-positive leukocytes outside the islets (21). Islet samples from non-diabetic healthy and balanced manage patients prove to minimal CXCL10 expression too as an absence of CXCR3-positive cell infiltration (21–23). CXCL10 expression in the vicinity of the islet is largely, however perhaps not exclusively, derived from β cells and/or islet-infiltrating leukocytes in the animal model (3, 16, 18, 24) too as in humans (8, 19). In summary, the pattern of islet CXCL10 expression and resulting CXCR3 positive T-cell infiltration in humans generally appears to be mirrored in the NOD model; however, the histopathology of infiltrating cells and resulting insulitis do differ in Essential ways, and insulitis is not necessarily present in at-risk people prior to the onset of TIDM, as it is in the pre-diabetic NOD mice (2).
