|
| Status |
Public on Nov 25, 2018 |
| Title |
EyeBank_cultured_choroid_stroma_fibroblast_cells_001_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
primary choroidal fibroblast cell population
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: eye
cell type: choroidal stromal fibroblasts
age: 80
Sex: Male
|
| Growth protocol |
Fibroblats and RPE cells were expanded in the same culture medium : Dulbecco-Vogt Modified Eagle Medium supplemented with 10% fetal bovine serum and antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
isolation protocol: RPE sheets were dislodged from the choroid after an ezymatic digestion (0.02% EDTA or 2U/ml dispase), rinsed and seeded for cell expansion. Choroidal fibroblats were obtained after treatment with 0.125 U/ml collagenase H.
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of passage 2 cells cultivated 6 days
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Nov 24, 2018 |
| Last update date |
Nov 25, 2018 |
| Contact name |
Stéphanie Proulx |
| Organization name |
Université Laval
|
| Street address |
1050 Chemin Ste-Foy
|
| City |
Québec |
| State/province |
Qc |
| ZIP/Postal code |
G1S4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE122885 |
Gene expression of cultured human choroidal stromal fibroblasts and retinal pigment epithelial (RPE) cells |
|
