Extraction of high-quality epidermal RNA after ammonium thiocyanate-induced dermo-epidermal separation of 4 mm human skin biopsies.
To obtain a separation of the epidermal and dermal compartments to examine compartment specific biological mechanisms in the skin, we incubated 4 mm human skin punch biopsies in ammonium thiocyanate. We wanted to test (i) the histological quality of the dermo-epidermal separation obtained by different incubation times; (ii) the amount and quality of extractable epidermal RNA and (iii) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30-min incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally), but also varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments, and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately.
Clemmensen
Anders
A
Department of Dermatology, Odense University Hospital, Odense, Denmark. anders.clemmensen@ouh.regionsyddanmark.dk
Thomassen
Mads
M
Clemmensen
Ole
O
Tan
Qihua
Q
Kruse
Torben A
TA
Petersen
Thomas K
TK
Andersen
Flemming
F
Andersen
Klaus E
KE
D001706:Biopsy / Q000295:instrumentation* / Q000379:methods*; D016000:Cluster Analysis; D020405:Dermis / Q000187:drug effects*; D004817:Epidermis / Q000187:drug effects*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D012867:Skin / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D013861:Thiocyanates / Q000494:pharmacology*; D014158:Transcription, Genetic
D013861:Thiocyanates; D012313:RNA; C026976:thiocyanic acid