|
| Status |
Public on Mar 01, 2011 |
| Title |
In vitro exp 3 LNCaP 50µM ABR-215050 24h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Experimental samples after 24h treatment.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
|
| Treatment protocol |
Cell cultures were exposed to ± 50µM ABR-215050 for 24h at 37°C. After 24h the cell culture medium was removed and the adherent cells were washed with 10 ml cold PBS before 1 ml Trizol® (Invitrogen) was added. After homogenization the Trizol was transferred into 2 ml Eppendorf tubes and frozen at -70°C.
|
| Growth protocol |
LNCaP cells were seeded at a cell density of 1-106 cells per 5 ml culture medium (RPMI-10% FCS) 25 ml cell culture flasks the day before treatment started.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was isolated by extracting the Trizol® (Invitrogen) samples with chloroform followed by separation and purification using a modified protocol for the RNeasy RNA extraction Kit (Qiagen). 1 µl RNAse inhibitor mix was added per 50 µl total RNA before treatment with DNAse for 20 min (DNA-free™; Ambion, Austin TX). The RNA concentration and purity was quality controlled by analysis with a Bioanalyser.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Fluorescently labeled cDNA targets for hybridization were prepared according to manufacturers' instructions using the Corning Pronto Plus system 6 (Corning).
|
| |
|
| Channel 2 |
| Source name |
Control samples no treatment.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
|
| Treatment protocol |
Cell cultures were exposed to ± 50µM ABR-215050 for 24h at 37°C. After 24h the cell culture medium was removed and the adherent cells were washed with 10 ml cold PBS before 1 ml Trizol® (Invitrogen) was added. After homogenization the Trizol was transferred into 2 ml Eppendorf tubes and frozen at -70°C.
|
| Growth protocol |
LNCaP cells were seeded at a cell density of 1-106 cells per 5 ml culture medium (RPMI-10% FCS) 25 ml cell culture flasks the day before treatment started.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was isolated by extracting the Trizol® (Invitrogen) samples with chloroform followed by separation and purification using a modified protocol for the RNeasy RNA extraction Kit (Qiagen). 1 µl RNAse inhibitor mix was added per 50 µl total RNA before treatment with DNAse for 20 min (DNA-free™; Ambion, Austin TX). The RNA concentration and purity was quality controlled by analysis with a Bioanalyser.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Fluorescently labeled cDNA targets for hybridization were prepared according to manufacturers' instructions using the Corning Pronto Plus system 6 (Corning).
|
| |
|
| |
| Hybridization protocol |
Prior to hybridization, arrays were UV-cross-linked at 500 mJ/cm2 and pretreated using the Universal Microarray Hybridization Kit (Corning) according to manufacturers' instructions. Slides were hybridized using Corning Pronto Plus system 6 (Corning) on a MAUI®Hybridization System.
|
| Scan protocol |
Arrays were washed using Universal Microarray Hybridization Kit (Corning) according to manufacturers' instructions and fluorescence was recorded using an Agilent G2565AA microarray scanner (Agilent Technologies).
|
| Description |
na
|
| Data processing |
TIFF images were analyzed using the Gene Pix Pro software (Axon Instruments, Foster City, CA), and the quantified data matrix was loaded into a local installation of BioArray Software Environment (BASE) (http://base.thep.lu.se). Quantified intensities for treated sample were stored as channel 1 (ch1) whereas quantified intensities for control sample were stored as channel 2 (ch2). Median spot pixel values were used to calculate background corrected intensities (Int1 and Int2). Spots that were flagged during image-analysis or had a background corrected intensity value >64999 signal units were removed and regarded as missing values. Spots with a background corrected intensity value <1 signal units were set to value 1. Spots from dye-swap hybridization were merged using geometric mean of ratios requiring presence in both replicates. For spots, log ratio (M) was calculated as log2(Int1/Int2) and average intensity (A) was calculated as log10(Int1*Int2)/2. Normalization was performed by applying Lowess on M values stratified into 8 separate groups defined spatially by pin-tip (8 neighboring pin-tips per group).
|
| |
|
| Submission date |
Jul 09, 2009 |
| Last update date |
Mar 01, 2011 |
| Contact name |
Johan Vallon-Christersson |
| Organization name |
Lund University
|
| Department |
Department of Oncology, Clinical Sciences
|
| Street address |
Lund University
|
| City |
Lund |
| ZIP/Postal code |
SE-221 85 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL5886 |
| Series (1) |
| GSE17031 |
Tasquinimod, a Quinoline-3-Carboxamide Anti-Angiogenic Agent, treated Prostate Cancer Cell Line in-vivo and in-vitro |
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