|
| Status |
Public on Jun 03, 2010 |
| Title |
Subject 219, Replicate 4 |
| Sample type |
RNA |
| |
|
| Source name |
Derived from neonatal foreskin
|
| Organism |
Homo sapiens |
| Characteristics |
subject/donor: 219
gender: Male
tissue: cultured epidermis
replicate: 4
|
| Treatment protocol |
Following overnight incubation in MMM Media, individual epidermal tissues were peeled from the tissue insert and placed into 2 ml of RNAlater® (Ambion/ABI, Foster City, CA) pre-loaded into a 24-well plate. After 24 hours incubation under 4°C, the RNAlater® submersed tissues were transferred into a 2 ml Seal-Rite® microcentrifuge tube (USA Scientific Inc, Ocala, FL) and all excess RNAlater® was removed from the tissue by gentle pipetting. The tissues were stored at -80°C until RNA was isolated.
|
| Growth protocol |
Reconstructed human EpiDerm™ EPI-200 tissues prepared from four different neonatal foreskin donors (254, 1188, 219 and 926) were acquired from MatTek Corporation (Ashland, MA). Five tissues from each donor were placed into 6-well plates with 1ml/tissue/well of pre-warmed (37°C) NMM media (MatTek Corp.) and incubated overnight at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRI-Reagent and protocol provided by supplier (Molecular Research Center, Cincinnati, OH). Extracted RNA was further purified using Qiagen RNEasy columns and protocol provided by supplier (QIAgen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Total RNA was converted to biotin-labeled cRNA GeneChip target using Ambion Message Amp II- Biotin Enhanced Kit according to manufacture's recommendations. Biotin-labeled cRNA was purified using the Qiagen RNeasy kit and a total of 20 μg of cRNA was fragmented randomly to ~200 bp at 94°C for 35 min (200 mM Tris-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc).
|
| |
|
| Hybridization protocol |
Following fragmentation, cRNA was hybridized for 16 hr at 45C on GeneChip Human U133plus2.0 Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from reconstituted human epidermis
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
|
| |
|
| Submission date |
Dec 04, 2009 |
| Last update date |
Jun 02, 2010 |
| Contact name |
Jay Patrick Tiesman |
| Organization name |
Procter & Gamble
|
| Department |
Global Biotechnology
|
| Lab |
Mason Business Center
|
| Street address |
PO BOX 8006
|
| City |
MASON |
| State/province |
OH |
| ZIP/Postal code |
45040-8006 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE19330 |
Expression data from Epiderm cultured skin derived from four different donors |
|
