|
Status |
Public on Apr 01, 2009 |
Title |
NDMA biological replicate 3 |
Sample type |
RNA |
|
|
Channel 1 |
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 |
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.
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|
|
Channel 2 |
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 |
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 3 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.
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|
|
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 |
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