Rapid, high-quality and epidermal-specific isolation of RNA from human skin.
As global transcriptome analyses with a growing demand on layer-specific applications are widely used in cutaneous biology, we investigated the effect of established and optimized dermo-epidermal separation methods on the quality of RNA. We compared enzymatic separation with dispase, chemical separation with 1 M sodium chloride and heat separation to a treatment with 3.8% ammonium thioyanate. The impact of freezing as well as the addition of 10 mM aurintricarboxylic acid was considered in the evaluation of the amount and quality of isolated RNA from dermis and epidermis. Using the low abundant gene kallikrein 12 for real-time PCR analysis, we were able to demonstrate the superior RNA quality after dermo-epidermal separation using 3.8% ammonium thiocyanate. In addition to the time effectiveness this separation technique promises dermal and epidermal purity and is therefore the method of choice for producing high-quality RNA for genome-wide dermal and epidermal transcriptional analysis.
Trost
Andrea
A
Division of Molecular Dermatology, Department of Dermatology, Paracelsus Private Medical University Salzburg, Salzburg, Austria. a.trost@salk.at
Bauer
Johann W
JW
Lanschützer
Christoph
C
Laimer
Martin
M
Emberger
Michael
M
Hintner
Helmut
H
Onder
Kamil
K
D002469:Cell Separation; D010450:Endopeptidases / Q000494:pharmacology; D004817:Epidermis / Q000737:chemistry*; D019085:Fluorescent Antibody Technique, Direct; D052816:Foreskin / Q000737:chemistry; D005615:Freezing; D006358:Hot Temperature; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification; D012965:Sodium Chloride / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D012333:RNA, Messenger; D013861:Thiocyanates; D012965:Sodium Chloride; D012313:RNA; C026976:thiocyanic acid; D010450:Endopeptidases; C497170:KLK12 protein, human; D007610:Kallikreins; C023139:dispase