Epigenetic Analysis of Laser Capture Microdissected Fetal Epithelia1

• Published in: Analytical biochemistry Vol. 442; no. 1; pp. 68 - 74
• Main Authors: Seelan, Ratnam S., Warner, Dennis R., Mukhopadhyay, Partha M., Andres, Sarah A., Smolenkova, Irina A., Wittliff, James L., Pisano, M. Michele, Greene, Robert M.
• Format: Journal Article
• Language: English
• Published: 30-07-2013
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2013.07.029

Laser capture microdissection (LCM) is a superior method for non-destructive collection of specific cell populations from tissue sections. While 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 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 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 miRNA expression levels and CpG methylation.