|
| Status |
Public on Oct 20, 2013 |
| Title |
Dermal papilla cells-spheroid culture-donor D5 |
| Sample type |
RNA |
| |
|
| Source name |
Cultured cell dermal papilla spheroids-passage 3
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
donor id: D5
tissue origin: Occipital Scalp
tissue/cell type: Cultured cell dermal papilla spheroids
passage: p3
|
| Treatment protocol |
Cells were cultured in dMEM containing 10% FBS. No antibiotics were used during cell culture. Cells were passaged using Trypsin-EDTA. Cells were fed every four days, and when RNA was collected, it was done so 72 hours post feeding cells that were 70% confluent. For spheroids, RNA was collected 48 hours after hanging drop cultures were started.
|
| Growth protocol |
Intact papillae were isolated from occipital scalp skin from 3 male donors undergoing hair transplantation procedures. 27G needles were used for microdissection. For culture, isolated papillae were adhered in 35mm dishes, at which point they collapsed, and cells migrate out from the papillae forming an explant (passage 0). Spheroids containing 3000 cells were established from cells cultured at passage 3, using hanging drop culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells and tissues were collected in RLT buffer containing BME. Total RNA was isolated using the mRNA micro Kit from Qiagen.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix two-cycle amplification kit was used to generate biotin labelled cRNA. Two rounds of amplification were performed, following the manufacturers instructions.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15ug cRNA was hybridized onto U133 Plus 2.0 genechips from Affymetrix. The Affymetrix GeneChip Hybridization Oven 640, and the GeneChip Fluidics Station 450 were used for hybridization and chip processing.
|
| Scan protocol |
Chips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from dermal papilla spheroids
DP16
|
| Data processing |
Data was analyzed using GeneSpring 12.0 software. Data was RMA normalized, baseline transformed (baseline to median of all samples), and log2 transformed for analysis within GeneSpring. We used a one way ANOVA, coupled with a Benjamini-Hochberg multiple testing correction to perform paired comparisons between cells at different stages in culture respective to intact papilla. Other analytical software was also employed. We used iPAGE (RMA normalized values used here), and GEDI (RMA normalized, baseline and log2 transformed values used) to identify pathways and metagenes with interesting changes in gene expression.
|
| |
|
| Submission date |
Mar 01, 2013 |
| Last update date |
Oct 20, 2013 |
| Contact name |
Claire A Higgins |
| E-mail(s) |
ch2609@columbia.edu
|
| Organization name |
Columbia University
|
| Department |
Department of Dermatology
|
| Lab |
Christiano Lab
|
| Street address |
1150 St Nicholas Avenue, Russ Berrie 307
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE44765 |
Global profiling of human hair follicle scalp dermal papilla cells using Affymetrix microarrays |
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