Seelan Ratnam S, Warner Dennis R, Mukhopadhyay Partha M, Andres Sarah A, Smolenkova Irina A, Wittliff James L, Michele Pisano M, Greene Robert M
Birth Defects Center, Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville, Louisville, KY 40202, USA.
Anal Biochem. 2013 Nov 1;442(1):68-74. doi: 10.1016/j.ab.2013.07.029. Epub 2013 Jul 30.
Laser capture microdissection (LCM) is a superior method for nondestructive collection of specific cell populations from tissue sections. Although DNA, RNA, and protein have been analyzed from LCM-procured samples, epigenetic analyses, particularly of fetal, highly hydrated tissue, have not been attempted. A standardized protocol with quality assurance measures was established to procure cells by LCM of the medial edge epithelia (MEE) of the fetal palatal processes for isolation of intact microRNA for expression analyses and genomic DNA (gDNA) for CpG methylation analyses. MicroRNA preparations, obtained using the RNAqueous Micro kit (Life Technologies), exhibited better yields and higher quality than those obtained using the Arcturus PicoPure RNA Isolation kit (Life Technologies). The approach was validated using real-time polymerase chain reaction (PCR) to determine expression of selected microRNAs (miR-99a and miR-200b) and pyrosequencing to determine CpG methylation status of selected genes (Aph1a and Dkk4) in the MEE. These studies describe an optimized approach for employing LCM of epithelial cells from fresh frozen fetal tissue that enables quantitative analyses of microRNA expression levels and CpG methylation.
激光捕获显微切割（LCM）是从组织切片中无损收集特定细胞群体的一种优越方法。虽然已对LCM获取的样本进行了DNA、RNA和蛋白质分析，但尚未尝试对表观遗传学进行分析，尤其是对胎儿的高度水合组织。建立了一个带有质量保证措施的标准化方案，通过对胎儿腭突内侧边缘上皮（MEE）进行LCM来获取细胞，以分离用于表达分析的完整微小RNA和用于CpG甲基化分析的基因组DNA（gDNA）。使用RNAqueous Micro试剂盒（赛默飞世尔科技公司）获得的微小RNA制剂，与使用Arcturus PicoPure RNA分离试剂盒（赛默飞世尔科技公司）获得的制剂相比，产量更高、质量更好。该方法通过实时聚合酶链反应（PCR）验证，以确定所选微小RNA（miR-99a和miR-200b）的表达，并通过焦磷酸测序确定MEE中所选基因（Aph1a和Dkk4）的CpG甲基化状态。这些研究描述了一种优化方法，用于对新鲜冷冻胎儿组织的上皮细胞进行LCM，从而能够对微小RNA表达水平和CpG甲基化进行定量分析。
