Epigenetic analysis of laser capture microdissected fetal epithelia

Abstract

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.

Differential transmission of the molecular signature of RBSP3, LIMD1 and CDC25A in basal/ parabasal versus spinous of normal epithelium during head and neck tumorigenesis: A mechanistic study

“…To the best of our knowledge, reports indicating the reason behind varying expression of the genes in different layers of normal oral epithelium are unavailable. Moreover, the technique of using LCM for separately analyzing promoter methylation in different layers of normal oral epithelium has not been reported, although it was used for RNA expression or epigenetic alterations in other organs [ 13 , 34 ]. Interestingly, no promoter methylation of the genes was observed in the composite normal epithelium, although its low frequency was previously reported [ 35 ].…”

Spatiotemporal Expression and Functional Analysis of miRNA-22 in the Developing Secondary Palate

“…Increased expression of a particular miRNA is, thus, usually coupled with decreased expression of its target mRNA(s), and vice versa . As such, numerous miRNAs have been shown to be important regulators of differentiation during embryogenesis in general (Du et al, 2016), and orofacial development in particular (Mukhopadhyay et al, 2010; Sheehy et al, 2010; Nie et al, 2011; Barritt et al, 2012; Seelan et al, 2013; Warner et al, 2015). Many miRNAs, such as miR-140, miR-200b, miR-133b, miR-146a, and the miR-17-92 cluster, have been associated with an increased risk for CP in humans and in animal models (Ries et al, 2017; Schoen et al, 2017; Wang et al, 2017; Pan et al, 2018; Suzuki et al, 2018).…”