|
| Status |
Public on Apr 27, 2016 |
| Title |
NNK_7d_2 |
| Sample type |
RNA |
| |
|
| Source name |
liver, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone , 7 days, 20 mg/kg
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Crl:WI[Gl/BRL/Han]IGS BR
tissue: liver
treatment_short_name: NNK
treatment_full_name: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
group: NNK_7d
control_group: CMC_1F_7d
treatment_duration: 7 days
treatment_dose: 20 mg/kg
|
| Treatment protocol |
Animals (8–10 weeks old) were assigned to dose groups (five rats/group) by weight using a weight stratification-based computer program. Substances were administered by gastric gavage for up to 14 days (in a volume of 5 ml/kg body weight/day) based on the group mean weekly body weight for each dose group.
|
| Growth protocol |
Male Wistar Hanover rats (Crl:WI[Gl/BRL/Han]IGS BR) from Charles River Laboratories, Inc. (Raleigh, NC) were maintained on certified rodent chow (Certified Rodent Diet 5200; Purina Mills, St. Louis, MO) ad libitum in individual suspended stainless steel wire-mesh cages. The animals were kept under controlled temperature (18–26°C), humidity (30–70%), and lighting (12 h light/dark cycle) and were acclimated for a minimum of 6 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen liver sections were grinded in a Mixer Mill MM 200 (Retsch GmbH and Co. KG, Haan, Germany) using precooled stainless steel balls. After adjusting the volume to 1ml with RLT lysis buffer, total RNA was isolated using Qiagen RNAeasy spin column kits, including a DNase digestion step according to the manufacturer’s instructions. A volume of lysate corresponding to 7mg of tissue was loaded RNeasy spin column. The final product was quality controlled by gel analysis using RNA 6000 Nano chips on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples with a peak area ratio >2.0 of 28S to 18S rRNA were used. RNA concentrations were determined with Ribogreen (Molecular Probes, Eugene, OR).
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled cRNA samples for hybridization on Affymetrix GeneChip RAE230A arrays were prepared according to the protocol supplied with the GeneChip Sample Cleanup module (Affymetrix Inc., Santa Clara, CA).
|
| |
|
| Hybridization protocol |
Starting with 5 μg of totalRNA with a 28S/18S rRNA peak ratio >1.7, biotin-labeled cRNA samples for hybridization on Affymetrix GeneChip RAE230A arrays were prepared accord- ing to the manufacturer’s instructions (Affymetrix, USA; GeneChip® Expression Analysis 701194 Rev.1). This protocol includes the use of the SuperScript Choice System (Invitro- gen Life Technologies) for cDNA synthesis and the ENZO RNA Transcript Labeling Kit (Affymetrix Inc.) for in vitro transcription. The cRNA (15 μg) was then fragmented in buffer supplied with the Cleanup Module and hybridized for 16 h at 45°C. The microarrays were washed and stained with streptavidin-phycoerythrin (SAPE, Molecular Probes) on the Affymetrix Fluidics Station 400 including an amplification step according to the manufacturer’s instructions.
|
| Scan protocol |
The fluorescent images of the GeneChips were captured with the Affymetrix GeneChip Scanner 3000.
|
| Data processing |
The raw microarray data was normalized using the RMA method implemented in R/Bioconductor.
|
| |
|
| Submission date |
Apr 21, 2015 |
| Last update date |
Apr 27, 2016 |
| Contact name |
Jonathan Moggs |
| E-mail(s) |
jonathan.moggs@novartis.com
|
| Organization name |
Novartis
|
| Street address |
Fabrikstrasse 2
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL341 |
| Series (2) |
| GSE68110 |
Trancriptional profiling of rat liver after short-term (up tp 14 days) administration of carcinogenic and non-carcinogenic chemicals |
| GSE68387 |
IMI MARCAR Project: towards novel biomarkers for cancer risk assessment |
|
