|
| Status |
Public on Dec 01, 2012 |
| Title |
NormalSkin_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Normal skin epithelium
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
breed: New Zealand White rabbit
gender: female
tissue: normal skin epithelium
|
| Treatment protocol |
The superficial incisional grids on rabbit ear were created by using a double blade scalpel with 1 mm space between the two blades (Fig.1A and 1B). A single layer of Tegaderm was used to cover the wounding areas on both right and left ears until the wounding areas were fully re-epithelialized (two days post wounding, POE0). Thereafter, the Tegaderm on right ear was completely removed (NOW) while four more layers of Tegaderms were applied on left ear wounds (FOW). Rabbit ear samples were harvested at POE0, POE3 and POE5.
|
| Growth protocol |
One half of the each harvested wound was incubated with 0.5M ammonium thiocyanate at room temperature for 30 min. The epidermis of the wounding area was separated from the dermis and rinsed with PBS solution.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The epidermis was immediately put into Tri Reagent® for RNA isolation (Sigma-Aldrich, St. Louis, MO) following the manufacture instruction. The TurboTM DNase (Life Technologies, Carlsberg, CA) was used to remove the genomic DNA from the total RNA followed by a RNA clean-up step with RNeasy® Mini Kit (Qiagen, Valencia, CA).
|
| Label |
Cy3
|
| Label protocol |
RNA concentration was determined on a NanoDrop Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) and sample quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). cDNA was synthesized from 400 ng of total RNA using the Quick-Amp labeling Kit (Agilent Technologies, Inc.) according the manufacturer’s protocol. Briefly, the cDNA was used as a template for in vitro transcription (IVT) in the presence of T7 RNA Polymerase and cyanine labeled CTP’s. The amplified, labeled mRNA was purified using the RNeasy Mini Kit (Qiagen).
|
| |
|
| Hybridization protocol |
For each array, 1650 ng of Cy3 labeled was fragmented and hybridized with rotation at 65°C for 17 hours. Samples were hybridized to rabbit custom design 4X44k arrays (Agilent Technologies, Inc).
|
| Scan protocol |
The arrays were washed according to the manufacturer’s protocol and then scanned on an Agilent G2505B scanner. Data were extracted using Feature Extraction 10.1.1.1 software (Agilent Technologies, Inc.).
|
| Data processing |
Using Limma software package in R environment (R386 2.15.2), the background of extracted microarray data was corrected by ‘normexp’ with offset 50. After normalized within and between arrays, the data was fit in a linear model with the empirical Bayes to moderate the standard errors. The p values of output data were adjusted with Benjamini and Hochberg's method to control the false discovery rate.
|
| |
|
| Submission date |
Nov 30, 2012 |
| Last update date |
Dec 01, 2012 |
| Contact name |
Wei Xu |
| E-mail(s) |
wei-xu@northwestern.edu
|
| Phone |
312 908 0566
|
| Organization name |
Northwestern University, Feinberg School of Medicine
|
| Department |
Surgery
|
| Lab |
Laboratory for Wound Repair and Regenerative Medicine
|
| Street address |
303 E Chicago Ave, Tarry 4-714
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL11170 |
| Series (1) |
| GSE42653 |
Expression of proinflammatory genes in epidermal keratinocytes is regulated by the hydration status. |
|
