Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA114849 | ENA |
REPOSITORIES: ENA
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Project description: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.
2010-03-03 | GSE15101 | GEO
Project description: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. 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).
2010-03-03 | E-GEOD-15101 | biostudies-arrayexpress
Project description:In an RNAseq analysis, we have identified the HOXC13-AS significantly downregulated in wound biopsies and epidermal cells compared to skin counterparts. To study the genes regulated by HOXC13-AS, we transfected HOXC13-AS siRNA pool into human primary epidermal keratinocytes to knockdown HOXC13-AS and induced cell differentiation for 3 days by 1.5 mM calcium. We performed a global transcriptome analysis of keratinocytes upon the HOXC13-AS knockdown using Affymetrix arrays.
2022-06-15 | GSE206103 | GEO
Project description:A total of 3 patients with basal cell carcinoma (BCC) and 3 healthy individuals (control; non-lesional skin) were enrolled in the study. Punch biopsies (4 mm) were obtained under local anaesthesia and immediately put in RNAlater (Qiagen, Hilden, Germany) and stored at - 80 °C until RNA extraction.
2016-03-29 | GSE74858 | GEO
Project description:We used 100 mg of stem and cell-culture induced callus tissues for the protein extraction. After protein quant, quality control, protein digestion and high pH RP separation, we performed the DIA proteomics of these samples.
2022-05-30 | PXD034226 |
Project description:We show that exposure of artificial human skin tissue to intense, picosecond-duration THz pulses affects expression levels of numerous genes associated with non-melanoma skin cancers, psoriasis and atopic dermatitis. Genes affected by intense THz pulses include nearly half of the epidermal differentiation complex (EDC) members. EDC genes, which are mapped to the chromosomal human region 1q21, encode for proteins that partake in epidermal differentiation and are often overexpressed in conditions such as psoriasis and skin cancer. In nearly all the genes differentially expressed by exposure to intense THz pulses, the induced changes in transcription levels are opposite to disease-related changes. Total RNA from exposed artificial human skin tissues to picosecond-duration broadband (0.2–2.5 THz) THz pulses with 1 kHz repetition rate, 1/e2 spot-size diameter of 1.5 mm and pulse energies of either 1.0 ?J or 0.1 ?J. For comparison, we have exposed skin tissues to UVA pulses (400 nm, 0.1 ps, 0.024 ?J).
2013-07-09 | E-GEOD-48586 | biostudies-arrayexpress
Project description:Psoriasis is a chronic inflammatory skin disease characterized by marked proliferation of keratinocytes leading to pronounced epidermal hyperplasia, elongation of rete ridges and hyperkeratosis. The most common form of psoriasis, chronic plaque psoriasis (Psoriasis vulgaris), involves relatively stable occurrence and progression of sharply demarcated lesions, usually on the trunk and extremities, which share a combination of trademark histological features, including tortuous and dilated dermal capillaries, loss of the epidermal granular layer, and accumulation of neutrophils beneath parakeratotic scale. In this study, whole-genome transcriptional profiling was used to characterize gene expression in 4 lesional and uninvolved skin samples obtained from patients with stable chronic plaque psoriasis. Skin mRNA expression was analysed by microarray. Four individuals with chronic plaque psoriasis were enrolled. 6 mm punch biopsies were obtained under local anaesthesia (lidocaine) from uninvolved skin and a target plaque.
2013-12-10 | E-GEOD-50790 | biostudies-arrayexpress
Project description: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.
2010-08-31 | GSE22022 | GEO
Project description:Human skin fibroblasts from an individual with hereditary vitamin D-resistant rickets bearing a homozygous p.Arg30* VDR mutation [Damiani et al., Osteoporos Int 2015; 26(6):1819-23] and from an age/sex-matched control were obtained from 4-mm punch biopsies of the forearm skin, after institutional board approval and with informed consent. Skin explants were fragmented in 6-well tissue culture plates and covered in complete AmnioMAX™ C-100 medium until attachment to surface; after approximately 12 days fibroblasts grown out of explants covered well surfaces completely. Secondary fibroblast cultures were maintained in high glucose DMEM supplemented with 10% FBS and 1% P/S. Fibroblasts were used for experiments between passages five to fifteen. Global gene expression of CO and MUT fibroblasts in response to 1,25D or ethanol vehicle (Veh) was analysed using microarrays. Six independent biological replicates were performed for each experimental condition: CO Veh, CO 1,25D, MUT Veh and MUT 1,25D. Cells were grown in 6-well plates and treated with 10 nM 1,25D or ethanol (1 ul/ml of medium) for 24 hours before RNA extraction. Based on quality control of extracted RNA performed with the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), four samples of each condition were chosen for microarray gene expression analysis, all with RNA integrity number above 8.00. Samples were processed according to manufacturer’s instructions, starting with 200 ng total RNA. Altogether, sixteen samples of labeled fragmented cDNA were hybridized to GeneChip Human Gene 2.0 ST Arrays (Affymetrix). Array data was analysed using Partek Genomics Suite, and based on quality control one array (MUT_Veh_4) was excluded. A Benjamini-Hochberg-corrected p-value cut-off of 0.05 was used for selecting significant differentially expressed genes.
2019-02-28 | GSE127314 | GEO
Project description:Human in vivo skin wound: Non-wounded skin was obtained by taking punch biopsies from three healthy donors (donor 1,2 and 3). The samples were termed 'skin day 0 in vivo wound'. Skin wound samples were retrieved by making new punch biopsies from the edge of the original biopsies after four days. These samples were termed 'skin day 4 in vivo wound'. As much dermal tissue as possible was removed by dissection to make sure mainly epidermis was present in the samples. The samples were washed in NaCl to possible remove infiltrating inflammatory cells before RNA isolation. Ex vivo skin wounds: Skin was obtained from three healthy donors following reduction surgery (donor 1, 2, and 3). As much dermal tissue as possible was removed dissection. These samples were termed 'skin day 0 ex vivo wound'. Skin was sliced into 1x10 mm slices and incubated in keratinocyte medium for four days with either 1:1000 fold dilution of DMSO or 10 micromolar AG-1478 (dissolved in DMSO). Again as much dermal tissue was removed by dissection as possible before RNA was isolated. These samples were termed 'skin day 4 ex vivo wound' and 'skin day 4 AG-1478 ex vivo wound'. By comparing the gene expression day 4 in ex vivo wound with in vivo wounds it was possible to see which part of the gene expression in wounded skin that was due to the epidermal reaction to injury and how much was due to stimuli from infiltrating inflammatory cells absent in the ex vivo skin wounds. By comparing the data from ex vivo skin wounds day 4 with and without the EGFR-inhibitor AG-1478, it was possible to look at the importance of the EGF-receptor of EGFR for the gene expression in ex vivo wounded skin.
2011-09-13 | E-MEXP-3305 | biostudies-arrayexpress