Laser Capture Microdissection Tailored to Type 1 Diabetes Mellitus Research

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 use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like 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 with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

Comparison of Acute Gene Expression Profiles of Islet Cells Obtained via Laser Capture Microdissection between Alloxan- and Streptozotocin-treated Rats

“…Transcriptomic analysis using high-quality RNA from frozen islets isolated via laser capture microdissection provided unique information about gene expression in the pancreatic islets of ALX- and STZ-treated rats. RIN values (mean: 7.23) in this study were similar to those from previous reports using RNA isolated from rodent islets by means of microdissection (Butler et al 2016; Szulawski et al 2016). Moreover, the RIN values reported here align with RNA data isolated via microdissection from organ/tissues other than pancreatic islets (Boone et al 2015; Catlin, Huse, and Boekelheide 2014; Torres Mena et al 2014).…”

“…animal (Tonne et al 2013). Further, the use of costly reagents is minimized, and multiple samples can be processed in a single day when using the laser capture microdissection method (Szulawski et al 2016).…”

“…The extremely high RNase content of the exocrine pancreas necessitates care in sample processing (Bynum and Ronzio 1976; Erickson et al 2009); therefore, little is known about islet gene expression profiles in these toxicity models. Recently, laser capture microdissection was used to easily isolate islet RNA of sufficient quantity and quality for use in an expression study (Szulawski et al 2016). Unlike the collagenase perfusion method of isolating pancreatic islets, laser capture microdissection allows both pathological and gene expression analyses to be performed with the same animal (Tonne et al 2013).…”

“…Unlike the collagenase perfusion method of isolating pancreatic islets, laser capture microdissection allows both pathological and gene expression analyses to be performed with the same animal (Tonne et al 2013). Further, the use of costly reagents is minimized, and multiple samples can be processed in a single day when using the laser capture microdissection method (Szulawski et al 2016).…”

Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4–Induced Type 1 Diabetes in Female Nonobese Diabetic Mice

“…Slides were air dried, followed by brief incubation on a hot plate and immediately taken to the Leica LMD6000 microscope (Buffalo Grove, Ill) for islet isolation as previously described. 37 …”