|
| Status |
Public on Oct 21, 2016 |
| Title |
Fibroblasts, sham, parallel to repeated UVR, rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Primary neonatal skin fibroblasts, sham with repeated UVR, replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary neonatal skin fibroblasts
gender: male
|
| Treatment protocol |
Fibroblasts were washed twice by PBS, then irradiated through a thin cover of PBS with UVA or UVB.
|
| Growth protocol |
Primary human fibroblasts were established from neonatal foreskin, and were incubated in Dulbecco's Modified Eagle's Medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37℃ with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacture's protocol. RNA was quantified using a NanoDrop-1000 spectrophotometer and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
Each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method using Arraystar Flash RNA Labeling Kit (Arraystar, Rockville, MD).
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA (pmol Cy3/μg cRNA) was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed and fixed.
|
| Scan protocol |
The hybridized array was scanned with using the Agilent DNA Microarray Scanner G2505C using one color scan setting for 8*60K array slides.
|
| Description |
Gene expression in fibroblasts harvested parallel to samples with three daily UV irradiations
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the scanned images.
Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent). LncRNAs were further analyzed when their expression was detectable in at least 6 out of 18 samples/ in at least one third of the samples.
|
| |
|
| Submission date |
Oct 20, 2016 |
| Last update date |
Oct 21, 2016 |
| Contact name |
Kazuyuki Yo |
| E-mail(s) |
ka-you@pola.co.jp
|
| Phone |
+81458267232
|
| Organization name |
POLA Chemical Industries, Inc.
|
| Department |
Skin Research Department
|
| Street address |
560 Kashio-cho, Totsuka-ku
|
| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
244-0812 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE89005 |
Changes of lncRNA expression in primary human skin fibroblasts after irradiation with UVA or UVB |
|
