|
| Status |
Public on Jan 05, 2016 |
| Title |
No treatment |
| Sample type |
RNA |
| |
|
| Source name |
human corneal epithelial cell
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: human corneal epithelial model
|
| Treatment protocol |
The NIR device emits an NIR spectrum between 1100 and 1800 nm, with water filtering to remove wavelengths between 1400 and 1500 nm, and simulates solar NIR radiation that reaches the human tissues on the Earth’s surface. To avoid thermal effects, the sapphire contact cooling tip was set to a fixed temperature of 20°C. We performed 5 rounds of NIR irradiation at 10 J ⁄cm2.
|
| Growth protocol |
Human corneal cells were prepared from normal human corneal epithelial tissue by enzymatic digestion, which was supplied from Rocky Mountain Lyons Eye Bank (CO, USA) after concluding the material transfer agreement. The medium was changed every 2 days until the cultures became subconfluented. The subconfluented corneal cells were subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with 0.4 μm pore size. Corneal cells were cultivated between an air and liquid interface to form a multilayered, highly corneal epithelium-like structure. All cells were grown at 37°C in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted in QIAzol reagent (Qiagen, Hilden, Germany) and spin-column purified using RNeasy Mini spin column (Qiagen). The RNA was extracted from control and irradiated group, which consists of six whole individual 3-dimensional reconstructed human corneal epithelial models. Quantity and quality of RNA samples were then measured using UV absorbance (NanoDrop Technologies, Wilmington, DE) and quality was also assessed using an Agilent 2100 bioAnalyzer series II (Agilent Technologies, Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA samples were synthesized using an Agilent Low Input Quick Amp Labeling kit, according to the manufacturer's instructions (Agilent Technologies). For each time point, 50 ng of total RNA was used to generate first-strand cDNA. After the denaturation step (10 min at 65°C) and cRNA synthesis step (2 hr at 40°C), the reactions were incubated at 70°C for 15 minutes to inactivate the AffinityScript enzyme. For the labeling reactions, cRNA samples were each mixed with 6 μL of Transcription Master Mix cocktail containing Cy3-CTP, and incubated at 40°C for 2 hours. Purification was performed using Qiagen RNeasy mini spin columns (Qiagen). Labeled cRNA were quantified on a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE) and quality was also checked by Agilent 2100 bioAnalyzer series II (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Hybridization mixtures were prepared by adding 2x Hybridization Buffer and mixing well by pipetting. Hybridization was carried out at 65°C for 17 hours. Washing was performed using the Agilent Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned using an Agilent Technologies Microarray Scanner (Agilent Technologies), and images were processed by Agilent Feature Extraction software (Agilent Technologies) with background correction.
|
| Description |
Gene expression of no treatment control
|
| Data processing |
Data were imported into GeneSpring and normalization was performed 75 percentile shift. After normalization and data filtering of the raw date output files, log-fold changes in gene expression in NIR irradiated group compared with control were analyzed to determine up-regulated and down-regulated genes, using a 2-fold change as the cut-off using GeneSpring GX 11.0 software (Agilent Technologies). Up-regulated and down-regulated genes were further clustered into functional gene groups using ontology analysis with GeneSpring GX 11.0 software.
|
| |
|
| Submission date |
Jan 04, 2016 |
| Last update date |
Jan 05, 2016 |
| Contact name |
Yohei Tanaka |
| E-mail(s) |
info@clinicatanaka.jp
|
| Phone |
+81-80-4335-0016
|
| Organization name |
Clinica Tanaka
|
| Department |
Plastic Surgery
|
| Street address |
M-1 Bld 1F, 3-4-3, Ote
|
| City |
Matsumoto |
| State/province |
Nagano |
| ZIP/Postal code |
3900874 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE76500 |
Gene expression in a 3-dimensional reconstructed human corneal epithelial tissue after near-infrared irradiation simulating solar near-infrared radiation that reaches human tissues on the Earth’s surface |
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