|
| Status |
Public on Jan 12, 2011 |
| Title |
CD200+CD49+Repeat2 |
| Sample type |
RNA |
| |
|
| Source name |
CD200+CD49+
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Haired Scalp
sex: male
age: 30-60s
|
| Treatment protocol |
Cells were not treated
|
| Growth protocol |
Cells were not expanded but isolated enzymatically from biopsied human tissue
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAeasy Qiagen protocol
|
| Label |
biotin
|
| Label protocol |
Briefly, 5ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to U133A Affymetrix microarray for human and 430 v2 for mouse. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
| Description |
Gene expression data from FACS sorted human adult keratinocytes using cell surface markers
|
| Data processing |
Affymetrix GeneChip Operating System (GCOS, v. 1.4, Affymetrix, Inc.) was used to quantitate expression signal levels for the arrays; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. These values were exported as .cel files. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. The flag values were additionally exported in .chp files.
Affymetrix probe intensities were imported into GeneSpring (v7.2, Agilent Technologies) where probeset signal values were calculated using the GC-RMA algorithm. Upon import of the Affymetrix flag values, the probesets were filtered to retain only those that were flagged P (present) in at least two of the repeat samples. This list was used for condition-based principle components analysis (PCA) to assess global trends in sample similarity. This analysis demonstrated groupings based on sample identity and prompted the use of mixed-model 2-way ANOVA as a means of finding differentially regulated genes between the sites of interest.
GC-RMA signal data was imported into Partek Genomics Solution (PGS, v6.2, Partek, Inc.), where the data were log2 transformed. Probesets not shared between mouse and human chips were removed. Genes less than p<0.05 in p-values for the identity of sample term of the ANOVA, and less than 2 fold changes between positive and negative populations were also excluded. The resulting enriched gene lists for bulge, secondary hair germ, and CD200highITGA6high cells were then examined for intersection using Partek.
|
| |
|
| Submission date |
Apr 28, 2010 |
| Last update date |
Jan 12, 2011 |
| Contact name |
Luis Andres Garza |
| E-mail(s) |
LAG@jhmi.edu
|
| Phone |
410-955-8662
|
| Organization name |
Johns Hopkins School of Medicine
|
| Department |
Dermatology
|
| Lab |
Garza lab
|
| Street address |
1550 Orleans St. Suite 204 CRBII Koch
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE21567 |
Human CD200+CD49+ hair follicle keratinocytes versus CD200-CD49+ keratinocytes |
| GSE21569 |
Bald scalp retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells |
|
