Female (age range 33-49), skin biopsy from upper nates. No nickel exposure
Treatment protocol
A total of 3 patch tests with 5% nickel sulfate was taken from each subject: i) the first patch test was exposed for 7h and a skin biopsy was taken immediately after removing the patch test ii) the second patch test was exposed for 48h immediately followed by a skin biopsy and iii) the third patch test was exposed for 48h and the skin biopsy was taken 48h after removing the patch test (the 96h biopsy). In addition, all participants were exposed to an empty patch test exposed for 48h followed by a skin biopsy.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, the skin biopses were placed in a morter filled with liquid nitrogen and ground mechanically using a pistil. The tissue was transferred to a lysis buffer and RNA was extracted using the RNase Lipid Tissue Kit (Qiagen, Valencia; CA). The amount and quality of the extracted RNA was evaluated using a lab-on-a-chip Bioanalyzer (Agilent Technologies, CA, USA).
Label
Phycoerythrin
Label protocol
First strand cDNA was synthesized from 1 ug total RNA by incubation (42C, 1 hr) in a 20 ml reaction volume containing 2.5 mM T7-(dT)24 primer, 50 mM Tris-HCl pH 8.3, 75 mM KCL, 3 mM MgCl2, 10 mM DTT and 500 mM dNTP, 10 U/ml Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA).Second strand cDNA was synthesized directly by adding 91 ml RNase free water, 30 ml 5 times second strand reaction buffer (Invitrogen), 3 ml 10 mM dNTP, 1 ml Escherichia coli DNA ligase (10 U/ml), 4 ml E. coli DNA polymerase I (10 U/ml), 1 ml E. coli RNase H (2 U/ml) followed by incubation (2 hr, 16C). The ends of the double-stranded cDNA were polished using T4 DNA polymerase (20 U, 5 min, 16C). The cDNA was purified and concentrated by phenol/ chloroform extraction and ethanol precipitation.
Hybridization protocol
The Affymetrix GeneChip® Hybridization, Wash, and Stain kit was used according to the protocol supplied with the kit.
Scan protocol
The Affymetrix system was used and the protocols supplied by Affymetrix followed.
Description
The sample is a part of an analysis of gene expression time-course in the human skin during elicitation of allergic contact dermatitis
Data processing
Log(2) transformed expression measures were calculated with the RMA procedure using the software from www.Bioconductor.org
