|
| Status |
Public on Jun 09, 2010 |
| Title |
Kidney H2O-treated 03 days replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Hupki mouse kidney
|
| Organism |
Mus musculus |
| Characteristics |
tissue: kidney
time point: 3 days
agent: H2O
|
| Treatment protocol |
Female Hupki mice (2-4 months old) were randomly assigned to dose groups (three animals per time point) and were treated daily with 5 mg/kg body weight aristolochic acid I (AAI) by gavage according to a protocol published previously [Mengs, U. (1988) Tumour induction in mice following exposure to aristolochic acid. Arch Toxicol, 61, 504-505] for 3, 12 or 21 days, respectively. Control animals were treated with vehicle, water only. Mice were killed after 24 h. All animal experiments were carried out under license in accordance with the law, and following local ethical review. Organ sections (kidney and liver) were collected, snap-frozen in liquid nitrogen and stored at -80ºC until further analysis.
|
| Growth protocol |
Hupki mice (Trp53 tm1/Holl, homozygous for the knock-in TP53 allele harbouring human TP53 sequences in the 129/Sv background) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and breed at the Institute of Cancer Research Animal Facility.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol (Invitrogen) following manufacturer's instructions and purified by Qiagen RNAease mini kit following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
5 μg of RNA was mixed with 1.2 μl T7 promotor primer (Agilent low RNA input linear amplification kit) and denatured at 65°C for 10 min. After being incubated on ice for 5 min, 8.5 μl cDNA Master Mix (Agilent) was added and the mixture was incubated at at 40°C for 2 hours. The sample was incubated at 65°C for 15 min and placed on ice for 5 min. Then the sample was mixed with Transcription Master Mix (Agilent) and Cy5 (tissue RNA) or Cy3 (UMRR) according to manufature's instuctions. After incubation at 40°C for 2 hours, the sample was purified with RNeasy Mini kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
universal mouse reference RNA
|
| Organism |
Mus musculus |
| Characteristics |
reference: 11 mouse cell lines collection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol (Invitrogen) following manufacturer's instructions and purified by Qiagen RNAease mini kit following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
5 μg of RNA was mixed with 1.2 μl T7 promotor primer (Agilent low RNA input linear amplification kit) and denatured at 65°C for 10 min. After being incubated on ice for 5 min, 8.5 μl cDNA Master Mix (Agilent) was added and the mixture was incubated at at 40°C for 2 hours. The sample was incubated at 65°C for 15 min and placed on ice for 5 min. Then the sample was mixed with Transcription Master Mix (Agilent) and Cy5 (tissue RNA) or Cy3 (UMRR) according to manufature's instuctions. After incubation at 40°C for 2 hours, the sample was purified with RNeasy Mini kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Test sample (Cy5-labelled) and reference (Cy3-labelled) were mixed and hybridised with Gene Expression Hybridization Kit (Agilent) on Agilent whole genome mouse 44k arrays following manufacturer's instructions. The arrays were incuated in Agilent SureHyb-enabled hybridization chambers, which was placed in the oven for 18 hours ( 65°C; 10 rpm).
|
| Scan protocol |
After hybridisation, the arrays were washed by Agilent Gene Expression Wash Buffer following the instructions. The arrays were scanned with a Axon 4000B scanner at wavelength 635nm and 532nm.
|
| Description |
KICON03DG14C5
|
| Data processing |
The images were analysed with BlueFuse software to extract expression values, then all data were loaded into GeneSpring V7.2 software. The data were LOWESS normalised and natural log-ratios were used for t-test and other analyses. Normalised ratios were used for fold-change analysis.
|
| |
|
| Submission date |
Sep 24, 2009 |
| Last update date |
Jun 09, 2010 |
| Contact name |
Ian Giddings |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
+44 20 8722 4293
|
| Fax |
+44 20 8722 4141
|
| URL |
http://www.crukdmf.icr.ac.uk
|
| Organization name |
Institute of Cancer Research
|
| Department |
Section of Molecular Carcinogenesis
|
| Lab |
CANCER RESEARCH UK DNA Microarray Facility
|
| Street address |
15 Cotswold Road
|
| City |
Sutton |
| State/province |
Surrey |
| ZIP/Postal code |
SM2 5NG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE18246 |
Gene expression changes induced by the human aristolochic acid I in renal and hepatic tissue of mice |
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