|
| Status |
Public on Oct 01, 2009 |
| Title |
Monocytes_10 µM T0901317 48h_donor1 |
| Sample type |
RNA |
| |
|
| Source name |
Monocytes from donor1 treated for 48h with 10µM T0901317
|
| Organism |
Homo sapiens |
| Characteristics |
Monocytes were isolated from healthy donor1 with inform consent and treated in vitro with 10µM T0901317 for 48h
|
| Biomaterial provider |
Etablissement Français du Sang (EFS)
|
| Treatment protocol |
Human peripheral-blood monocytes were obtained from the Etablissement Français du Sang (Besançon, France). Briefly, mononuclear cells were isolated by Ficoll gradient centrifugation and monocyte negative selection was performed via magnetic activated cell sorting using the Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
|
| Growth protocol |
Cells were grown in RPMI 1640 medium with glutamax-I (Invitrogen, Paisley, UK) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Invitrogen) in an atmosphere of 95% air and 5% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Human total RNA was isolated with the use of Trizol (Sigma-Aldrich). RNA quality and integrity were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy3
|
| Label protocol |
Sample labeling was performed as detailed in the “One-Color Microarray-Based Gene Expression Analysis protocol (version 5.5, part number G4140-90040). Briefly, 1 µg of each total RNA samples was used for the amplification and labeling step using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) in the presence of cyanine 3-CTP (Perkin Elmer). Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
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| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the the “One-Color Microarray-Based Gene Expression Analysis protocol (version 5.5, part number G4140-90040) using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65 µg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Following hybridization, the microarrays were washed once with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with preheated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files.
|
| Description |
All the experiments from RNA extraction to data processing were performed by Miltenyi Biotec Microarray Service (Cologne)
|
| Data processing |
For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware).
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| |
|
| Submission date |
Oct 29, 2008 |
| Last update date |
Oct 31, 2008 |
| Contact name |
Cédric REBE |
| E-mail(s) |
[email protected]
|
| Organization name |
IFR100
|
| Department |
Hopital du Bocage
|
| Lab |
batiment B2
|
| Street address |
2 av marechal de Lattre de Tassigny
|
| City |
DIJON |
| ZIP/Postal code |
21000 |
| Country |
France |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE13407 |
Effect of T0901317, AM580 or T0901317+AM580 on monocyte gene expression |
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