Laser capture microdissection tailored to type 1 diabetes mellitus research.
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.
Department of Specialty Medicine, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Diabetes Institute, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.
Robert
R
Szulawski
Office of Research and Grants, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.
Masato
M
Nakazawa
Department of Specialty Medicine, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Diabetes Institute, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Department of Biological Sciences, Ohio University College of Arts & Sciences, Athens, OH.|Molecular & Cellular Biology Program, Ohio University College of Arts & Sciences, Athens, OH.|Biomedical Engineering Program, Ohio University Russ College of Engineering & Technology, Athens, OH.
Kelly D
KD
McCall
Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Molecular & Cellular Biology Program, Ohio University College of Arts & Sciences, Athens, OH.
Calvin B L
CB
James
Department of Specialty Medicine, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Diabetes Institute, Ohio University Heritage College of Osteopathic Medicine, Athens, OH.|Biomedical Engineering Program, Ohio University Russ College of Engineering & Technology, Athens, OH.
Frank L
FL
Schwartz
D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics* / Q000473:pathology*; D005260:Female; D007515:Islets of Langerhans / Q000378:metabolism / Q000473:pathology*; D061346:Laser Capture Microdissection / Q000379:methods*; D051379:Mice; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D012106:Research / Q000295:instrumentation*
D012313:RNA
RNA isolation; cresyl violet; cryostat; laser capture microdissection (LCM); non-obese diabetic mouse (NOD); toll-like receptor (TLR); type 1 diabetes mellitus (TIDM)