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Epigenetic analysis of laser capture microdissected fetal epithelia

Seelan, Ratnam S., Warner, Dennis R., Mukhopadhyay, Partha M., Andres, Sarah A., Smolenkova, Irina A., Wittliff, James L., Michele Pisano, M., Greene, Robert M.
Analytical biochemistry 2013 v.442 no.1 pp. 68-74
DNA, DNA methylation, epigenetics, epithelial cells, genes, microRNA, nondestructive methods, population, quality control, quantitative analysis, quantitative polymerase chain reaction, sequence analysis, yields
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.