|
| Status |
Public on Dec 12, 2019 |
| Title |
EyeBank_cultured_human_corneal_epithelial_cells_006 |
| Sample type |
RNA |
| |
|
| Source name |
primary human epithelial corneal cell population
|
| Organism |
Homo sapiens |
| Characteristics |
age: 71
Sex: female
protocol: cultivated 14 days without feeder layer
|
| Treatment protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Growth protocol |
Once the Descement membrane has been peeled, remaining corneas (stromas with their attached epithelial cells) are then incubated with dispase (2.5 mg/ml) overnight at 4°C. Using forceps, the epithelium is mechanically separated from the underlying stroma and treated with trypsin, which allows the human corneal epithelial cells (hCECs) to dissociate one from another. Once isolated, hCECs are seeded in tissues culture flasks together with feeder layer for expansion.
Cells are grown in DH medium (Dulbecco–Vogt modification of Eagle's medium with Ham's F12 in a 3:1 ratio supplemented with 5% FetalClone II serum, 5 μg/ml of insulin, 0.4 μg/ml of hydrocortisone, 10 ng/ml of epidermal growth factor, 10−10 M of cholera toxin, 100 IU/ml of penicillin, and 25 μg/ml of gentamycin) until they reach 75-95% confluence.
|
| Extracted molecule |
total RNA |
| Extraction 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
|
| Label |
Cy3
|
| Label 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.
|
| |
|
| Hybridization 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%).
|
| Scan protocol |
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.
|
| Description |
Gene expression of passage 3 cells cultivated 14 days without feeder layer (80% confluence)
|
| Data processing |
gPixNormIQR : The normalized Inter-quartile range of all of the inlier pixels per feature. The range is computed independently in each channel.
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| |
|
| Submission date |
Dec 11, 2019 |
| Last update date |
Dec 12, 2019 |
| Contact name |
Gaëtan Le-Bel |
| E-mail(s) |
gaetan.lebel17@gmail.com
|
| Organization name |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
Local H2-00, Hôpital du Saint-Sacrement,1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Quebec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE141841 |
Gene expression of human corneal epithelial cells grown with irradiated human fibroblasts or irradiated mouse 3T3 feeder layer . |
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