Isolation of human epidermal layers by laser capture microdissection: Application to the analysis of gene expression by quantitative real-time PCR

摘要

We describe, for the first time, an efficient protocol based on laser capture microdissection (LCM) for the isolation of human epidermal layers for gene expression profiling using quantitative real-time PCR. Two areas enriched either in basal or granular layers were isolated by LCM. Skin biopsies were fixed in dry ice-cooled isopentane, cryosectioned and stained before the laser procedure. High-quality total RNA was extracted from each microdissected sample, which allowed the analysis of the spatial distribution of mRNA transcripts from 10 innate immunity-related genes within the epidermal layers. Using integrin alpha-6/integrin beta-4 and corneodesmosin/filaggrin-2 sets as gene markers for the basal and granular layers, respectively, we showed that Toll-like receptor 2, RNase 7, human beta-defensin-2 and -3, psoriasin and nucleotide-binding oligomerization domain 1 are upregulated in the suprabasal layer of normal human epidermis. Our protocol, which is based on the rapid isolation of epidermal layers, can be used to follow transcriptional processes in specific areas of the epidermis and is a very promising tool to use in the study of numerous aspects of dermatology.