Compared to the analyses of handpicked islets, the use of LCM with specific staining for target cells avoids contamination by neighboring cells and the confounding effects of cell trauma/ischemia, which leads to alterations in cellular protein and gene expression due to the harsh chemical and/or mechanical processes during manual isolation. This approach has been particularly useful to obtain small amounts of islet tissue to characterize the genetic profiles of both murine and human pancreas. Laser capture microdissection (LCM) has emerged as a widely used technique for the isolation of specific types of cells or a minimum amount of parenchyma for a variety of downstream analyses such as proteomic studies, RNA assays by microarray, or RNA sequencing. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels.
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