|
| Status |
Public on Mar 01, 2011 |
| Title |
HaCaT_GlcPAF_Block_B |
| Sample type |
RNA |
| |
|
| Source name |
HaCaT cells, with GlcPAF, 24h, replicate B
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HaCaT
agent: GlcPAF
|
| Treatment protocol |
The assay was performed in defined keratinocytes serum-free medium without growth supplement. Cells were treated with respectively 5 µM Ino-C2-PAF, GlcPAF and edelfosine or left untreated (control) 24 h.
|
| Growth protocol |
HaCaT cells were grown in RPMI medium supplemented with heat-inactivated fetal bovine serum (10 %), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamine (440 mg/l). One day prior to experimentation, cells were adapted to defined keratinocyte serum-free medium with growth supplement (including insulin, EGF, and FGF).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit in accordance to the manufacturer instructions (Qiagen Hilden, Germany). The integrity of RNA was verified by the presence of the 28S and 18S rRNA on agarose gels and an A260/280 ratio in the range of 1.9–2.1.
|
| Label |
Cy3
|
| Label protocol |
200 ng of total RNA were used for production of fluorescent (Cyanine-3) cRNA as described in the Agilent analysis instruction manual for One-Color Microarray-Based Gene Expression Analysis (Agilent Technologies, Palo Alto, USA). Amplified cRNA was purificated using RNAeasy mini spin columns and subsequently quantified using NanoDrop ND-1000 UV-VIS Spectrophotometer.
|
| |
|
| Hybridization protocol |
All samples were hybridized to Agilent whole human genome microarray kit 44K (G4112A) according to the manufacturer´s instructions (One-Color Microarray-Based Gene Expression Analysis; Version 5.7).
|
| Scan protocol |
Arrays were scanned with the use of a GenePix 4000A Scanner (Axon Instruments-Molecular Devices, Sunnyvale, USA).
|
| Description |
Gene expression after 24h in 5µM GlcPAF stimulated HaCaT cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (version 9.5.3) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Data files from Feature Extraction were imported into GeneSpring® GX software version 10 (Agilent Technologies) and analyzed using GeneSpring® default settings.
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| |
|
| Submission date |
Mar 10, 2010 |
| Last update date |
Mar 01, 2011 |
| Contact name |
Geo Semini |
| E-mail(s) |
geosemini@yahoo.com
|
| Organization name |
Charité - Universitätmedizin
|
| Department |
Institute for Biochemie
|
| Street address |
Oudenarderstrasse 16
|
| City |
Berlin |
| ZIP/Postal code |
13347 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE20722 |
Influence of alkyl-phospholipids on the gene expression profile of immortalized keratinocytes HaCaT |
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