|
| Status |
Public on Apr 11, 2016 |
| Title |
CEC-Nega1 |
| Sample type |
RNA |
| |
|
| Source name |
corneal epithelium cells_negative control
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: corneal epithelium cells
|
| Growth protocol |
Human peripheral corneal epithelial cells (CECs) were isolated from donor cornea, respectively, by treatment with 1000 protease units (PU)/ml of dispase (Dispase type Ⅱ; Godo Shusei) at 4 ºC overnight and TrypLE Express (Life Technologies) at 4 ºC for 30 minute. CECs were cultured in CEC medium, which consisted of Dulbecco’s modified Eagle’s medium and Ham’s F-12 media (1:1 mixture) (Life Technologies) containing B27-supplement (Life Technologies), Rho-kinase (ROCK) inhibitor (10 mM) (Nacalai Tesque), epidermal growth factor (10 ng/ml) (Life Technologies), Epigallocatechin gallate (SIGMA) and penicillin-streptomycin (50 IU/ml) (Nakalai Tesque), at 37 ºC in a 5% CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Kit (QIAGEN) and was assessed by Agilent 2100 Bioanalyzer and the RNA 6000 LabChip Kit (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Microarray experiments were performed according to the manufacturer’s instruction. Twenty to one hundred nanograms of total RNA was labelled with cyanine 3-CTP and hybridized in SurePrint G3 Human GE 8 x 60 K (Agilent Technologies).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6.0 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, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8 x 60 K allay for 17 hours at 65°C in a rotating 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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides.
|
| Description |
negative control CEC cell
|
| Data processing |
The array data were analyzed using GeneSpring software ver.13.0 (Agilent). Gene expression values were normalized by excluding low-signal intensity data and percentile shifts.
|
| |
|
| Submission date |
Mar 15, 2016 |
| Last update date |
Apr 11, 2016 |
| Contact name |
Koji Kitazawa |
| E-mail(s) |
kkitazaw@koto.kpu-m.ac.jp
|
| Organization name |
Kyoto Prefectural University of Medicine
|
| Department |
Ophthalmology
|
| Street address |
465 Kajiicho Kamigyoku
|
| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
601-0841 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14550 |
| Series (2) |
| GSE67823 |
Master transcription factors in corneal epithelial cells |
| GSE79251 |
Master transcription factors in corneal epithelial cells [OVOL2 Knock-down] |
|
