|
| Status |
Public on Dec 12, 2016 |
| Title |
SS_P3 cells_MitA_rep.2 |
| Sample type |
RNA |
| |
|
| Source name |
SS cells_MitA_24 h
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: Sézary Syndrome (SS) patient
gender: female
age (yrs): 77
cell type: peripheral blood tumor cells
tumor stage: III
treatment: ECPx89
% cd3+ in pbmc: 68
cd4+/cd8+ ratio: 8.7
|
| Treatment protocol |
Expanded cells were treated, or not, with SAHA (2.6 µM) and MitA (223 nM) for 24 h.
|
| Growth protocol |
PBMC were collected from 4 Sézary Syndrome (SS) patients and were put in culture without prior further purification because of the high percentage of T lymphocytes and the great majority of CD4+ among them. Cells were expanded for seven days in RPMI medium in the presence of IL-7 (10 ng/ml) and anti-CD28 (mAb ascites 1/400).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were recovered after drug treatment and RNA was extracted with Qiagen RNeasy® mini Kit. On column DNA digestion was done with Qiagen Rnase-free Dnase set.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200ng RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity > 6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides.
|
| Description |
P3_2_M
US83700202_252800415382_S01_GE1_107_Sep09_2_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20110325) to obtain background subtracted and spatially detrended Processed Signal intensities. All data were normalized by quantile normalization.
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| |
|
| Submission date |
Dec 12, 2014 |
| Last update date |
Dec 12, 2016 |
| Contact name |
CHELBI RABIE |
| Organization name |
CIML
|
| Street address |
Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, Case 906, 13288 Marseille cedex 9
|
| City |
MARSEILLE |
| ZIP/Postal code |
13009 |
| Country |
France |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE64119 |
Insights into the combined action of SAHA (vorinostat) and Mithramycin A in Sézary cells through gene expression profiling |
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