|
| Status |
Public on Apr 14, 2016 |
| Title |
Skin_fibroblasts_IL-1alpha/TGF-beta_2 |
| Sample type |
RNA |
| |
|
| Source name |
Addition of IL-1alpha/TGF-beta
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Skin
cell type: fibroblast
gender: female
additative: IL-1alpha/TGF-beta
|
| Treatment protocol |
Cells were seeded 250.000 cells per well in a 6-well plates a ≥24 hours incubation followed to allow the fibroblasts to attach to the bottom, then the medium was removed and each well washed twice with phosphate buffered saline (PBS) to eliminate all serum residues. Serum free medium was added and the cells were serum starved for another 24 hours. Cells were incubated with 0.5 ng/ml TGF-β , wells that were incubated with IL-1α were pre incubated with 0.2 ng/ml IL-1α 3h before TGF-β were added, both from Sigma-Aldrich (St. Louis, MO, USA). Cells were harvested after 16 hours.
|
| Growth protocol |
Skin fibroblasts were isolated from dermis explants. Fibroblast were seeded in Dulbecco’s Modified Eagle Medium (Invitrogen AB Lidingö, Sweden) supplemented with 10% fetal bovine serum (Invitrogen AB). The fibroblasts were incubated (37°C and 5% CO2) and cultured until confluence. Cultures were trypsinized, resuspended in cryomedium 50% Dulbecco’s Modified Eagle Medium, 10% dimethyl sulfoxide (Merck Schuchardt OHG Hohenbrunn Germany) and 40% fetal bovine serum, and frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fibroblasts were harvested for RNA isolation between passages 5 and 8. Total RNA was extracted using the RNeasy Plus purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of extracted RNA were measured using a Nano-Drop ND-1000 spectrophotometer (Nano-Drop Technology Inc., Wilmington, DE, USA) (OD260/280=1.9–2.1; OD260/230=1.7–2.0). Further analysis of RNA quality was performed according to the manufacturer’s guidelines with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Only samples with a RIN (RNA integrity number) value above 8.0 were used.
|
| Label |
Cy3
|
| Label protocol |
Synthesis and labeling of cRNA were performed with the Low Input Quick Amp Labeling one-color kit (Agilent Technologies). The cRNA was purified using RNeasy Mini kits, and concentration was measured with a spectrophotometer. Only samples with cRNA yield ≥0.825 μg and specific activity ≥6.0 pmol Cy3/μg cRNA were used.
|
| |
|
| Hybridization protocol |
A total of 600 ng of cyanine 3–labeled, linearly amplified cRNA was used for the hybridization. The cRNA was hybridized to the Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray in a G2545A hybridization oven (Agilent Technologies).
|
| Scan protocol |
The slides were scanned with the Agilent Microarray Scanner G2565AA (Agilent Technologies). Agilent Feature extraction software (version 10.7.3.1, Agilent Technologies) was used to compute expression values and to monitor hybridization quality. Only hybridizations meeting the quality standard as established in the feature extraction software were used for subsequent data analysis.
|
| Data processing |
The raw expression data were background corrected and quantile normalized as previously described (Smyth GK, Speed T. Normalization of cDNA microarray data. Methods. 2003;31(4):265–73.), using the packages marray and arrayQualityMetrics within the Bioconductor Software Collection for R.
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| |
|
| Submission date |
Jun 02, 2015 |
| Last update date |
Apr 15, 2016 |
| Contact name |
Anita Koskela von Sydow |
| Organization name |
Örebro University
|
| Street address |
Södra Grev Rosengatan 32
|
| City |
Örebro |
| ZIP/Postal code |
70362 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE69447 |
Whole-genome microarray analysis of human skin fibroblasts |
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