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Status |
Public on Oct 02, 2010 |
Title |
AAF_25µM_120h_Replicate1 |
Sample type |
RNA |
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Source name |
embryonic mouse fibroblasts Balb/c 3T3 cell line
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Organism |
Mus musculus |
Characteristics |
strain: BALB/c cell line: 3T3 cell line treatment: 2-Acetylaminoflourene (AAF) dose: 25µM time: 120h
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Treatment protocol |
Balb/c 3T3 cells were seeded at 200 cells in each 60 x 15 mm culture dish with 4 ml M10F, using six culture dishes for every treatment. When cells reached a confluence of 60-65%, the culture medium was removed and replaced with fresh medium containing all tested chemicals at a specific concentration and two time points (24 and 120h). First we treated the cells both 24h and 120h with concentrations reported in the literature (0.5µM BaP, 50µM DAT, 25µM AAF and 2µM MCA). In a second approach IC20 concentrations were investigated for each chemical at both time points. The concentrations determined for IC20 ranged from 1.5 µM BaP to 700 µM DAT for 24h of treatment, and from 0.1 µM BaP or MCA to 10µM AAF for 120h of treatment. Balb/c 3T3 cells treated with 0.75% DMSO alone were kept as controls.
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Growth protocol |
The Balb/c 3T3 cell line was obtained from Cytotest Cell Research GmbH (RCC), Roßdorf, Germany. Cells were harvested and used within 9-10 passages to perform the experiments. Cells were cultured with M10F containing MEM (minimum essential medium) + 10% FBS and incubated at 37 °C and 5% CO2.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA-extraction was performed with the RNeasy Mini Kit (RNeasy MidiKit Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s instruction. A standard quality control of the total RNA was performed using the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA).
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Label |
biotin
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Label protocol |
Total RNA (5µg) was used to generate biotin-labeled cRNA (10 µg) by means of GeneChip® One-Cycle cDNA Synthesis Kit and GeneChip® IVT Labeling Kit (Affymetrix). Sample processing was performed exactly as described by the microarray manufacturer (Affymetrix). Quality control of cRNA was performed using a Bioanalyzer (Agilent 2001 Biosizing, Agilent Technologies).
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Hybridization protocol |
Following fragmentation, labeled cRNA of each sample was hybridized to Affymetrix GeneChip® Mouse Genome - 430 2.0 arrays covering approximately 36,000 full-length mouse gene transcripts and stained according to the manufacturer's instructions.
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Scan protocol |
The arrays were scanned using the GeneChip® Scanner 3000.
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Description |
n/a
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Data processing |
Array data was normalized using scaling or per-chip normalization to adjust the total or average intensity of each array to be approximately the same (GCOS). The Scale factor (SF) was according to the Affymetrix recommended setting SF=1 and the TGT value was 500 and were used to generate a microarray quality control and data report. A T-test analysis was performed using Data Mining Tool (DMT) 3.0 from Affymetrix. A stringent comparison between normal and tumoral tissues was performed according to the consistency of gene expression changes. A t-test and a ranking analysis with up- and down-regulated genes were performed and only genes that exhibit a 100% concordance in the change direction with a p-value < 0.05 (t-test) and a fold change (FC) *2/FC*-2 were reported.
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Submission date |
Jun 07, 2010 |
Last update date |
Oct 02, 2010 |
Contact name |
Astrid Rohrbeck |
E-mail(s) |
rohrbeck.astrid@mh-hannover.de
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Phone |
0511-5322807
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Organization name |
Medizinische Hochschule Hannover
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Department |
Department of Toxicology
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Street address |
Carl-Neuberg-Straße 1
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City |
Hannover |
State/province |
Niedersachsen |
ZIP/Postal code |
30625 |
Country |
Germany |
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Platform ID |
GPL1261 |
Series (1) |
GSE22180 |
In vitro carcinogenicity testing with Balb/c 3T3 Cells treated with various chemical carcinogens |
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