|
| Status |
Public on Jun 15, 2016 |
| Title |
LCL6_control_21days |
| Sample type |
RNA |
| |
|
| Source name |
LCL6_control_21days
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: depressive disorder
cell line: LCL6
treatment: control
clinical treatment outcome: responder
|
| Treatment protocol |
LCLs were treated with 0.5µg/ml fluoxetine (dissolved in DMSO) for 21 days. MOCK treated control cultures were grown in parallel.
|
| Growth protocol |
LCLs were grown in RPMI medium supplemented with 15% heat-inactivated fetal bovine serum, antibiotics (100 µg/ml penicillin, 100 µg/ml streptomycin) and a final concentration of 4 mM L-glutamine. Medium exchanging was done every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the NucleoSpin® RNA Kit (Macherey-Nagel, Germany) according to the manufacturer instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng 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, Germany). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 0.025ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 0.025ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized 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 SureScan Microarray Scanner using one color scan setting .
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10 (Agilent) using default parameters (protocol GE1_1100 and Grid: 039494_D_F_20120411) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jun 15, 2016 |
| Last update date |
Jun 15, 2016 |
| Contact name |
Julia Carolin Stingl |
| Organization name |
Federal Institute for Drugs and Medical Devices
|
| Department |
Research
|
| Street address |
Kurt-Georg-Kiesinger Allee 3
|
| City |
Bonn |
| ZIP/Postal code |
53175 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE83386 |
Gene expression of Lymphoblastoid Cell Lines (LCLs) from Depressed Patients after in-vitro treatment with fluoxetine |
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