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| Status |
Public on May 11, 2010 |
| Title |
Uremic Diet vrs Control rats replicate 2 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
Rat Aortic valves_Normal Diet_9 weeks
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
sex: Female
feed: Normal Diet
feeding time: 9 weeks
|
| Treatment protocol |
Aortic valves samples were obtained from 3 groups: a Uremic-diet group, a reversibility group and a control group (n=7 each). The rats in the Uremic-diet and the reversibility group were fed exclusively with a uraemia induced high-adenine (0.75%), high-phosphate diet (1.5%). The control group received normal rat chow with no treatment. After 9 weeks, rats in the Uremic-diet group and in the control were anaesthetized using ketamine/xylazine and sacrificed; the rats in the reversibility group were kept alive for an additional 10 weeks, on a normal rat chow, and then sacrificed.
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| Growth protocol |
Sixty one Sprague-Dawley female rats, 8 weeks old, each weighing about 250 g, were used for the study. The protocol was approved by the Hebrew University Ethics Committee.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation: 1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min. Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation: 1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min.
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| Label |
Cy3
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| Label protocol |
One microgram of total RNA was amplified and labeled with a fluorescent dye (either Cy3 or Cy5), using the Low RNA Input Linear Amplification & Labeling kit (Agilent Technologies, Palo Alto, CA, USA). The amount and quality of the resulting labeled cRNA was measured using the ‘micro-array measurement mode’ of the Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA).
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| Channel 2 |
| Source name |
Rat Aortic valves_Uremic Diet_9 weeks
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
sex: Female
feed: Uremic diet
feeding time: 9 weeks
|
| Treatment protocol |
Aortic valves samples were obtained from 3 groups: a Uremic-diet group, a reversibility group and a control group (n=7 each). The rats in the Uremic-diet and the reversibility group were fed exclusively with a uraemia induced high-adenine (0.75%), high-phosphate diet (1.5%). The control group received normal rat chow with no treatment. After 9 weeks, rats in the Uremic-diet group and in the control were anaesthetized using ketamine/xylazine and sacrificed; the rats in the reversibility group were kept alive for an additional 10 weeks, on a normal rat chow, and then sacrificed.
|
| Growth protocol |
Sixty one Sprague-Dawley female rats, 8 weeks old, each weighing about 250 g, were used for the study. The protocol was approved by the Hebrew University Ethics Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation: 1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min. Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation: 1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min.
|
| Label |
Cy5
|
| Label protocol |
One microgram of total RNA was amplified and labeled with a fluorescent dye (either Cy3 or Cy5), using the Low RNA Input Linear Amplification & Labeling kit (Agilent Technologies, Palo Alto, CA, USA). The amount and quality of the resulting labeled cRNA was measured using the ‘micro-array measurement mode’ of the Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA).
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| Hybridization protocol |
Equal amounts of Cy3 or Cy5 labeled cRNA were hybridized to the 4 X 44K Agilent Whole Rat Genome Oligo-Microarray (Agilent Technologies, USA) for 17 hours at 60°c in an Agilent DNA-Microarray Hybridization Oven. The arrays were later washed using Gene Expression Wash Buffer Kit from Agilent.
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| Scan protocol |
The Microarray was scanned using the GenePix4000B Scanner (Axon) and the data was extracted from the resulting images using GenePix Pro 4.1 software (Axon).
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| Description |
Technical Replicate 2 of 2
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| Data processing |
The resultant files (gpr) produced by GenePix have been analyzed utilizing LIMMA package of the R software. The Cy5 and Cy3 intensities within each array were normalized using the print-tip locally weighted scatter plot smoothing (LOESS) function, while no background correction was applied. To identify differentially expressed genes, a parametric empirical Bayesian approach implemented in LIMMA was used. A moderated t test was performed in parallel, with the use of a false discovery rate correction for multiple testing. LIMMA calculated an emission intensity A value [A = (log2(Cy5) × Cy3)/2] where Cy5 and Cy3 are the normalized emission intensities of each feature. P value < 0.05 confidence level has been used to pinpoint those significantly differentiated genes. Genes had to have an A-value (average expression level for the gene across all arrays and channels) of more than 8.5, thus avoiding faint emissions.
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| Submission date |
May 10, 2010 |
| Last update date |
Nov 20, 2012 |
| Contact name |
Mony Shuvy |
| E-mail(s) |
monysh@gmail.com
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| Phone |
972-508573458
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| Organization name |
Hadassah Hospital
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| Department |
Cardiology
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| Street address |
Ein-Karem
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| City |
Jerusalem |
| ZIP/Postal code |
91120 |
| Country |
Israel |
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| Platform ID |
GPL16305 |
| Series (1) |
| GSE21771 |
Raloxifene Attenuates Gas6 and Apoptosis in Experimental Aortic Valve Disease in Renal Failure |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM542552.gpr.gz |
5.9 Mb |
(ftp)(http) |
GPR |
| Processed data provided as supplementary file |
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