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| Status |
Public on May 20, 2008 |
| Title |
Transcript Profiling of High Fat Diet induced Obesity-prone and Obesity-resistant Rats |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by array
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| Summary |
Rodents respond to chronic high fat diet in at least two ways: some of them may readily gain body weight and become obese (termed obesity-prone), and others may not (termed obesity-resistant). An integrated approach of transcript and metabolic profiling of obesity-prone and obesity-resistant rats has been conducted, showing significantly different transcript and metabolic profiles in the two phenotypes. The major transcriptional differences involved hepatic fatty acid metabolism and ketogenesis in response to 16 weeks of high fat diet. At the same time, the different metabolic profiles (in liver tissue extracts, serum, and urine) between the two phenotypes could be ascribed to the corresponding pathways identified with multivariate statistical analysis, including fatty acid metabolism, Krebs cycle, and amino acid metabolism. The integration of results from both transcript and metabolic profiling revealed the different responses to dietary intervention of the two phenotypes and the physiological basis of susceptibility to metabolic disease in obesity-prone rats from a systematic view.
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| Overall design |
We developed OP and OR phenotypes in a group of Wistar rats by maintaining them on a HFD for 16 weeks, and subsequently analyzed their hepatic transcript profile using cDNA microarrays, the rats were feed on standard chow diet for 16 weeks as control. Four rats were selected for transcript analysis in each group.
The four control rats were mixed and labeled with cy5, each OP or OR rats were labeled with cy3.
Label protocol: 2 ug of total RNA were primed with T7 Promoter Primer (from the Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color) at 65 ℃ for 10 min, then reversed transcribed at 40℃ for 2 h in the presence of 1ul MMLV-RT (Agilent), and 10 mM dNTP, and RNase OUT (Agilent). cDNAs were amplified at 40℃for 2 hours in the presence of T7 RNA Polymerase (Agilent) and 4 ul 25mM aaUTP (Ambion).Generated cRNA was purified with RNeasy mini Kit (QIAGEN).Mix 4 ug amino allyl-modified cRNA and 100mg Cy3 NHS ester, incubate in the dark at room temperature (~25 ℃) for 1 hour then purified with RNeasy mini Kit (QIAGEN).
Hybridization protocol: After mixed with hybridization buffer (GE healthcare), samples were applied to microarray slide, cover the slip with care. Place the slide in a wet box.Incubate in a hybridization chamber at 42 ℃ for 16-18 hours, avoid light exposure. After hybridization, slides were washed sequentially with 1×SSC/0.2 SDS(50℃), 0.1×SSC/0.2%SDS(50 ℃) and 0.1×SSC(room temperature) before scanning.
Scan protocol: Axon GenePix 4000B
Description: Total RNA was prepared from rat livers using Trizol reagent (Invitrogen, USA), and further purified with an QIAGEN RNeasy Kit. Then, 2 ug of total RNA were amplified using Low RNA Input Linear Amplification Kit (Agilent, USA), and labeled with amino allyl-modified cRNA labeling kit (Ambion, USA). Equal amounts of the RNA from four control rats were mixed, and the mixture was labeled with Cy5, while the RNA from OP and OR rats was labeled with Cy3. The Cy5 and Cy3 probes were mixed and hybridized at 42 ℃ for 16 h. The two fluorescent images (Cy3 and Cy5) were scanned separately by Agilent Scanner G2565BA, and the image data were analyzed using ImaGene 4.2 (Biodiscovery, Santa Monica, CA, USA).
Data processing: LOWESS normalized, background subtracted VALUE data obtained from log of processed Green signal/processed Red signal (high fat diet rat/control rat).
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| Contributor(s) |
Li H, Xie Z, Wang Q, Zhu Z, Zhang Q, Jia W |
| Citation(s) |
19913842 |
| Submission date |
May 19, 2008 |
| Last update date |
Mar 19, 2012 |
| Contact name |
Zhang Qinghua |
| E-mail(s) |
qinghua_zhang@shbiochip.com
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| Organization name |
Shanghai shbiochip co
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| Street address |
151# libing road
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| City |
shanghai |
| ZIP/Postal code |
201203 |
| Country |
China |
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| Platforms (1) |
| GPL4294 |
SBC Rat cDNA Microarray For GBE50 v1.0 |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA106305 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE11492_RAW.tar |
14.2 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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