|
| Status |
Public on Apr 29, 2010 |
| Title |
Caco2_NDEA_6hr50mM_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control for NDEA 50mM 6h
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human colonic adenocarcinoma cells
cell line: Caco-2
|
| Treatment protocol |
Caco-2 cells were treated for 1, 6 or 24 hours with one of the following six NOC: MNNG (1μM), MNU (1mM), NDEA (50mM), NDMA (100mM), NPIP (40mM) and NPYR (100mM)
|
| Growth protocol |
Caco-2 cell cultures were transferred weekly by trypsinization and incubated at 37 ºC in a humidified incubator containing 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from TRIzol® suspended cells according to the manufacturer’s protocol with minor modifications, followed by a clean up, using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) with DNase treatment.
|
| Label |
Cy3
|
| Label protocol |
The Two-Color Microarray-Based Gene Expression Analysis kit from Agilent Technologies (Amstelveen, The Netherlands) was used to generate Cyanine (Cy) labelled cRNA according to the manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
NDEA 50mM 6h
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human colonic adenocarcinoma cells
cell line: Caco-2
|
| Treatment protocol |
Caco-2 cells were treated for 1, 6 or 24 hours with one of the following six NOC: MNNG (1μM), MNU (1mM), NDEA (50mM), NDMA (100mM), NPIP (40mM) and NPYR (100mM)
|
| Growth protocol |
Caco-2 cell cultures were transferred weekly by trypsinization and incubated at 37 ºC in a humidified incubator containing 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from TRIzol® suspended cells according to the manufacturer’s protocol with minor modifications, followed by a clean up, using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) with DNase treatment.
|
| Label |
Cy5
|
| Label protocol |
The Two-Color Microarray-Based Gene Expression Analysis kit from Agilent Technologies (Amstelveen, The Netherlands) was used to generate Cyanine (Cy) labelled cRNA according to the manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
Hybridization according to Agilent instructions.
|
| Scan protocol |
Slides were scanned on a GenePix® 4000B Microarray Scanner (Molecular Devices, Sunnyvale, USA). Cy3 and Cy5 were excited at wavelengths of 532 and 635 nm, respectively. Laser power was set to 100%. The photo multiplier tube gain was set to a saturation tolerance of 0.02% to minimize background and saturated spots. Photomultiplier gain was set to 587 (Cy3) and 705 (Cy5) for replicates 1 and 2, and 628 (Cy3) and 560 (Cy5) for replicates 3 and 4, based on automatic establishment of ideal scan settings. The images obtained (resolution 5 micron, 16 bit tiff image) were processed with Imagene 8.0.1 software (Biodiscovery, El Segundo, USA) to measure mean signal intensities for spots and local backgrounds followed by a quality control in Microsoft Excel.
|
| Description |
Biological replicate 1
|
| Data processing |
Data preparations were performed in GenePix Pro software (version 6.0, Molecular Devices) and included in chronological order: background subtraction, omission of bad spots (controls and irregularly shaped spots), and flagging of spots (control spots and bad spots). One raw data file was created by GenePix Pro (a GPR file) containing data from both channels (Cy3 and Cy5). LOWESS normalization, log (base=2) transformations and calculation of difference of test compound versus respective vehicle control (log2 ratios) were calculated in ArrayTrack (version 3.4).
|
| |
|
| Submission date |
Mar 22, 2010 |
| Last update date |
Apr 29, 2010 |
| Contact name |
Dennie Hebels |
| E-mail(s) |
d.hebels@maastrichtuniversity.nl
|
| Phone |
0031-43-3881088
|
| Fax |
0031-43-3884146
|
| URL |
http://www.grat.nl
|
| Organization name |
Maastricht University
|
| Department |
Health Risk Analysis and Toxicology
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| ZIP/Postal code |
6200MD |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE20993 |
Time-series toxicogenomics analysis of N-nitroso compound exposure in Caco-2 cells |
|
