Isolation of human epidermal layers by laser capture microdissection: application to the analysis of gene expression by quantitative real-time PCR.
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.
Glycobiology and Plant Extracellular Matrix (GlycoMEV) Laboratory, UPRES EA 4358, IFRMP 23, University of Rouen, Mont-Saint-Aignan, France.
Giuseppe
G
Percoco
Magalie
M
Bénard
Yasmina
Y
Ramdani
Elian
E
Lati
Luc
L
Lefeuvre
Azeddine
A
Driouich
Marie-Laure
ML
Follet-Gueye
D000328:Adult; D062506:Claudin-4 / Q000235:genetics; D051183:Desmoglein 1 / Q000235:genetics; D004817:Epidermis / Q000033:anatomy & histology / Q000378:metabolism*; D005260:Female; D000091344:Filaggrin Proteins; D015870:Gene Expression; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D039503:Integrin alpha6 / Q000235:genetics; D039663:Integrin beta4 / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins; D053547:Keratin-14 / Q000235:genetics; D061346:Laser Capture Microdissection; D008875:Middle Aged; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012260:Ribonucleases / Q000235:genetics; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins / Q000235:genetics; D051195:Toll-Like Receptor 2 / Q000235:genetics; D023083:beta-Defensins / Q000235:genetics
C109656:CDSN protein, human; D062506:Claudin-4; C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D051183:Desmoglein 1; C000717249:FLG protein, human; C000717250:FLG2 protein, human; D000091344:Filaggrin Proteins; D006023:Glycoproteins; D039503:Integrin alpha6; D039663:Integrin beta4; D036341:Intercellular Signaling Peptides and Proteins; D053547:Keratin-14; D012333:RNA, Messenger; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; C089242:S100A7 protein, human; D051195:Toll-Like Receptor 2; D023083:beta-Defensins; D012260:Ribonucleases; C470000:Ribonuclease 7