|
| Status |
Public on Mar 01, 2011 |
| Title |
In vivo exp 1 LNCaP 10mg kg ABR-215050 24h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Experimental samples after 24h treatment.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
|
| Treatment protocol |
Tumor bearing mice (2 animals on each time point) were exposed to ABR-215050 at 10 mg/kg for 24 h at day 14 and day 21 after inoculation before the tumors were excised, frozen at -70°C, and used for RNA extraction.
|
| Growth protocol |
Nude BALB/c mice (8-10 weeks old) were used for subcutaneous implantation of human prostate tumor cells LNCaP. Distribution for 24h of ABR-215050 at 10 mg/kg/day (administered orally via the drinking water (ad.lib.)) started on day 14 or day 21 after inoculation. All animal experiments were conducted in accordance with the Bioethics Committee guidelines in Lund, Sweden.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor samples were collected and stored at -70°C. About 50 mg of frozen tumor tissue were cut and cruched in LN2, followed by immediate addition of 1 ml Trizol® (Invitrogen). The total RNA was isolated by extracting the Trizol® 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 |
Tumor bearing mice (2 animals on each time point) were exposed to ABR-215050 at 10 mg/kg for 24 h at day 14 and day 21 after inoculation before the tumors were excised, frozen at -70°C, and used for RNA extraction.
|
| Growth protocol |
Nude BALB/c mice (8-10 weeks old) were used for subcutaneous implantation of human prostate tumor cells LNCaP. Distribution for 24h of ABR-215050 at 10 mg/kg/day (administered orally via the drinking water (ad.lib.)) started on day 14 or day 21 after inoculation. All animal experiments were conducted in accordance with the Bioethics Committee guidelines in Lund, Sweden.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor samples were collected and stored at -70°C. About 50 mg of frozen tumor tissue were cut and cruched in LN2, followed by immediate addition of 1 ml Trizol® (Invitrogen). The total RNA was isolated by extracting the Trizol® 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 |
GPL5186 |
| 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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