Adult human fibroblasts derived from the dermis of 4 mm punch biopsies taken from the lower backs of 15 healthy subjects of 3 different phototypes (types I, III and VI). This study was approved by the Human Research Ethics Committee of Hamburg. Subjects with type I skin were #4, 12, 15, 19 and 26; subjects with type III skin were # 6, 9, 13, 14 and 23, and subjects with type VI skin were # 7, 8, 16, 17 and 28. The fibroblasts were cultured from the biopsies and were grown in monolayer culture in high glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine plus 1% penicillin and streptomycin at 37°C in a humidified 5% CO2 atmosphere. Fibroblasts were subcultured using routine methods and were used at passages 3 to 6.
Overall design: Oligo-cDNA microarray hybridization was performed according to the National Cancer Institute in-house protocol, as detailed previously (Yamaguchi et al., 2008). Briefly, total RNAs were prepared from cultured fibroblasts using an RNeasy mini kit (Qiagen, Valencia, CA). The quality (purity and integrity) and quantity of each total RNA preparation was measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). A universal human reference RNA (Stratagene, La Jolla, CA) was used as a control. cDNA samples generated from RNA samples and purified before the coupling reaction were labeled with Cy3 (for fibroblast samples) or Cy5 (for reference RNA) mono-reactive dyes (GE Healthcare, Piscataway, NJ), and were hybridized simultaneously on an oligo-DNA chip (Hs-OperonV3.0-v1p24, p27, p31) overnight at 42°C. Two fluorescent intensities of the oligo-DNA chip were scanned using a microarray scanner (GenePix 4000B; Axon Instruments Inc., Molecular Devices Corp., Sunnyvale, CA). Differential gene expression was profiled using Genepix Pro 5.0 software and was analyzed by Miltenyi Biotec (Bergisch Gladbach, Germany).
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