使用激光捕获显微切割技术分析视网膜mRNA/miRNA表达及DNA甲基化
Use of laser capture microdissection for analysis of retinal mRNA/miRNA expression and DNA methylation.
作者信息
Hackler Laszlo, Masuda Tomohiro, Oliver Verity F, Merbs Shannath L, Zack Donald J
机构信息
Avidin Ltd, Szeged, Hungary.
出版信息
Methods Mol Biol. 2012;884:289-304. doi: 10.1007/978-1-61779-848-1_21.
Abstract
Laser capture microdissection (LCM) is a useful method to isolate specific cells or cell layers of interest from heterogeneous tissues, such as the retina. The collected cells can be used for DNA, RNA, or protein analysis. We have applied LCM technology to isolate cells from the outer nuclear, inner nuclear, and ganglion cell layers of the retina for mRNA and microRNA (miRNA) expression and epigenetic (DNA methylation) analysis. Here, we describe the methods we have employed for sample preparation, LCM-based isolation of retinal layers, RNA/DNA extraction, RNA quality check, microRNA analysis by quantitative PCR, and DNA methylation analysis by bisulfite sequencing.
摘要
激光捕获显微切割（LCM）是一种从异质性组织（如视网膜）中分离特定感兴趣细胞或细胞层的有用方法。收集到的细胞可用于DNA、RNA或蛋白质分析。我们已应用LCM技术从视网膜的外核层、内核层和神经节细胞层中分离细胞，用于mRNA和微小RNA（miRNA）表达以及表观遗传学（DNA甲基化）分析。在此，我们描述了我们用于样品制备、基于LCM的视网膜层分离、RNA/DNA提取、RNA质量检查、通过定量PCR进行的miRNA分析以及通过亚硫酸氢盐测序进行的DNA甲基化分析的方法。
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