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Series GSE15101 Query DataSets for GSE15101
Status Public on Mar 03, 2010
Title Extraction of high-quality epidermal RNA after NH4SCN induced dermo-epidermal separation of 4 mm human skin biopsies
Organism Homo sapiens
Experiment type Expression profiling by array
Summary To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but 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.
 
Overall design Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).
 
Contributor(s) Thomassen M, Clemmensen A
Citation(s) 19645824
Submission date Mar 04, 2009
Last update date Mar 25, 2019
Contact name Mads Thomassen
E-mail(s) [email protected]
Phone +4565412839
Fax +4565411911
Organization name Odense University Hospital
Department Clinical Genetics (KKA)
Street address Soender Boulevard 9
City Odense
ZIP/Postal code DK-5000
Country Denmark
 
Platforms (2)
GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
GPL571 [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Samples (37)
GSM377633 Subject1_buttock full skin_24h SLS_ammonium thiocyanate (matrix 1)
GSM377634 Subject1_buttock full skin_24h SLS_RNAlater (matrix 1)
GSM377635 Subject2_buttock full skin_24h SLS_ammonium thiocyanate (matrix 1)
Relations
BioProject PRJNA114849

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE15101_RAW.tar 109.0 Mb (http)(custom) TAR (of CEL)
Processed data included within Sample table
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