|
| Status |
Public on Feb 22, 2020 |
| Title |
Normal_fibrobalst_3_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Human primary fibroblast cells_cultured
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Korean primary fibroblasts
|
| Treatment protocol |
Cells were maintained in incubators at 37°C and exposed to normoxia (20% O2, 5% CO2)
|
| Growth protocol |
Fibroblast cultivation was performed using the dermal portion of keloid tissues obtained from 5 keloid patients. The specimens were cut into 2mm3 size and placed in 60mm culture dishes and were cultured in mixture of Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL) and Ham’s nutrient mixture F12 (Gibco BRL) medium, by a 3:1 ratio. The supplements included 10% fetal bovine serum (Hyclone), 100 U/ml of penicillin, 100 mg/ml streptomycin, 1×10-10 M cholera toxin, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 5 μg/ml transferrin, and 2×10-11 M triiodothyronine. The outgrown cells were harvested and the identities of cells were verified by vimentin and alpha-smooth muscle actin (α-SMA) expression. All primary cells used in this study were at passage-3. Cells were maintained in incubators at 37°C and exposed to normoxia (20% O2, 5% CO2) or hypoxia (1% O2, 5% CO2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, 10 cell lines (5 NFs and 5 KFs) were grown in replicate cultures and subjected to RNA extraction using the Qiagen RNeasy Mini Kit (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
100 ng of total RNA was used to prepare biotinylated aRNA (cRNA) according to the manufacturer’s protocol (30 IVT Express Kit, Affymetrix 901228)
|
| |
|
| Hybridization protocol |
Samples were prepared for hybridization using 10 µg of biotinylated aRNA in a 1X hybridization cocktail according the Affymetrix hybridization manual. Additional hybridization cocktail components were provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit. GeneChip arrays (Human PrimeView, Affymetrix 901837) were hybridized in a GeneChip Hybridization Oven at 45°C for 16hr at 60 RPM. Washing was done using a GeneChip Fluidics Station 450 per the manufacturer’s instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
|
| Scan protocol |
Images were extracted with Affymetrix GeneChip Command Console (AGCC)
|
| Description |
Gene expression data from Korean normal primary fibroblast cells
|
| Data processing |
Images were extracted with Affymetrix GeneChip Command Console (AGCC) and analyzed using GeneChip Expression Console. A Primeview CDF (provided by Affymetrix) that included probe information for the ERCC controls (GPL16043) was used to generate the CEL files.
|
| |
|
| Submission date |
Feb 21, 2020 |
| Last update date |
Feb 22, 2020 |
| Contact name |
Hensin Tsao |
| E-mail(s) |
HTSAO@mgh.harvard.edu
|
| Organization name |
massachusette general hospital
|
| Street address |
50 Blossom Street
|
| City |
boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL16043 |
| Series (1) |
| GSE145725 |
Hypoxia and HIF-1α Regulate Collagen Production in Keloids |
|
