Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray profiling of expression patterns of fibroblasts from different skin types

ABSTRACT: 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. 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Vincent Hearing

PROVIDER: E-GEOD-22022 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin.

Choi Wonseon W Wolber Rainer R Gerwat Wolfram W Mann Tobias T Batzer Jan J Smuda Christoph C Liu Hongfang H Kolbe Ludger L Hearing Vincent J VJ

Journal of cell science 20100824 Pt 18

Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used micro ...[more]

PMID: 20736300

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Project description:The data from these cell lines is described in figure 2 of the mansucript "Systemic and cell-type specific gene expression patterns in scleroderam skin" by Whitfield et al. (2003) PNAS. Aortic smooth muscle cells and microvascular endothelial cells were obtained from Clonetics and cultured in the recommended media; Hs578T myofibroblast-like cells were grown in RPMI-1640 supplemented with phenol red, glutamine (2 mM) and 5% fetal calf serum; HeLa S3 epithelial cells were obtained from the American Type Culture Collection (ATCC) and cultured as described in Whitfield et al., MBC, (2002). Normal, morphea and scleroderma fibroblasts were derived from normal, morphea and scleroderma skin biopsies taken by Dr. Kari Connolly at UCSF. In all cases, RNA was prepared from cells 48 hrs after changing the culture media to avoid the induction of serum responsive genes. Poly(A) RNA from each cell line was reverse-transcribed into Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA and reference RNA reverse-transcribed into Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA using standard methods. Cells grown in culture were analyzed on cDNA microarrays containing 40,512 elements representing 28,384 UniGene clusters (UniGene Build No. 155, released 9-28-2002) manufactured in the Stanford Microarray Facility (http://www.microarray.org). Equal amounts of Cy5- and Cy3-labeled cDNA were hybridized to spotted cDNA microarrays and scanned using a GenePix 4000A Scanner (Axon Instruments, Union City CA). Detailed protocols are available at http://brownlab.stanford.edu/protocols.html/. Data were extracted by superimposing a grid over each array using GenePix 3.0 software (Axon Instruments, Union City CA). Spots of poor quality, determined by visual inspection, were excluded from further analysis. A cell type comparison design experiment design type compares cells of different type for example different cell lines. Keywords: cell_type_comparison_design

Dataset Information

Microarray profiling of expression patterns of fibroblasts from different skin types

Publications

The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin.

Similar Datasets

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