A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection.
A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection.
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DOI：
10.3390/biom11050625
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发表时间：
2021-04-22
期刊：
影响因子：
5.5
通讯作者：
Kulkarni RN
中科院分区：
文献类型：
--
作者：
Cefalo CMA;Mezza T;Giaccari A;Kulkarni RN
从内分泌胰腺切片中分离高质量的RNA是一个相当大的挑战，这主要是由于高核糖核酸酶水平。激光捕获显微切割（LCM）的哺乳动物胰岛，与RNA提取协议，已成为一种可行的方法来表征其遗传和蛋白质组学的概况。然而，仍然缺乏从LCM衍生的人胰腺标本中获得适合于下一代测序分析的高质量RNA的经验证的方案。在这项研究中，我们应用了四种方法（Pisphure提取试剂盒，Qiazol方案，Qiazol + Clean-up试剂盒和RNeasy Microkit + Carrier）从非糖尿病个体和接受部分胰腺切除术的2型糖尿病患者获得的人类胰岛中提取RNA，以及从非糖尿病和糖尿病器官供体中挑选的胰岛。使用Nanodrop 100通过260/280吸光度和使用生物分析仪的RNA完整性数来确定总RNA的产率和纯度。结果表明，在四种方法中，RNeasy MicroKit + Carrier（Qiagen）提供了最高的产率和纯度。
The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. 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. 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. 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. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity.
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通讯作者：
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