|
| Status |
Public on Apr 01, 2009 |
| Title |
NDMA biological replicate 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NDMA 100mM 24h
|
| Organism |
Homo sapiens |
| Characteristics |
Caco-2 (Human colonic adenocarcinoma cells)
|
| Treatment protocol |
Caco-2 cells were treated for 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 |
Control for NDMA 24h
|
| Organism |
Homo sapiens |
| Characteristics |
Caco-2 (Human colonic adenocarcinoma cells)
|
| Treatment protocol |
Caco-2 cells were treated for 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 4 of 4
|
| Data processing |
Data preparations were performed in GeneSight software (BioDiscovery) and included in chronological order: background subtraction, omission of bad spots (controls and irregularly shaped spots), LOWESS normalization, log (base=2) transformations, calculation of difference of test compound versus respective vehicle control.
|
| |
|
| Submission date |
Jan 05, 2009 |
| Last update date |
Jan 05, 2009 |
| 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 |
GPL6480 |
| Series (1) |
| GSE14284 |
Molecular signatures of N-nitroso compounds in Caco-2 cells: implications for colon carcinogenesis |
|
