|
| Status |
Public on Apr 20, 2017 |
| Title |
Left ventricle_normal sodium_angiotensin II_replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Left ventricle, normal sodium, angitoensin II, replicate 3
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
tissue: heart left ventricle
gender: male
sodium: normal sodium
|
| Treatment protocol |
At the end of the experiment, heart left ventricles were collected in RNA later and frozen (-80°C) for further mRNA extraction
|
| Growth protocol |
Sprague-Dawley rats were fed for 8 weeks 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) (n=40 in each). After 4 weeks, rats were infused or not with angiotensin II (200 ng.kg-1.min-1, sc) for 4 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were dissociated in RLT buffer using a Fast-Prep® homogeneizer (MP Biomedicals). Total RNA was extracted using RNeasy® Fibrous Tissue mini-kit (Qiagen). RNA quality and integrity were determined using the Agilent 2100 Bioanalyser (Agilent Technologies).
|
| Label |
cy3
|
| Label protocol |
200ng of each total RNA samples was used for amplification and labelling with the Agilent Low Input Quick amp Labelling kit, one color. Yields of cRNA and dye incorporation rate were measured using ND-1000 spectrometer (NanoDrop technologies, Peqlab biotechnologies GmbH, Erlangen, Germany).
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| |
|
| Hybridization protocol |
Hybridization procedure was performed using Agilent Gene Expression Hybridization kit. Briefly, 1.65µg Cy3-labeled fragmented cRNA was hybridized 17h at 65°C to Agilent Rat Gene Expression Microarray V3 4x44K using recommended manufacturer’s hybridization chambers and oven. The microarrays were then washed once with the Agilent Gene Expression Wash Buffer for 1 min at RT, and then washed with Agilent Gene Expression Wash buffer 2 at 37°C for 1 min.
|
| Scan protocol |
Fluorescence signals were detected using Agilent’s Microarray Scanner System and the Agilent Feature Extraction Software (version 11.0.1.1) to read out and process the microarray image files.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Statistical analysis was performed using R software version 3.3.04 along with the Limma package. The method used for background correction was based on the normal-exponential convolution model with the saddle-point approximation to maximum likelihood. Normalization was performed using cyclic loess method. Only probes whose signal was considered as higher than background in at least four out of six replicates in at least one condition were selected for further analysis. Within-array replicate probes were replaced with their average. The assessment of differentially expressed mRNAs between NS diet and LS diet was performed using the limma GLM (Generalized Linear Model) method followed by Benjamini Hochberg correction for multiple testing. Genes with a corrected p-value lower than 0.1 were selected for further investigation.
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| |
|
| Submission date |
Jul 18, 2016 |
| Last update date |
Apr 21, 2017 |
| Contact name |
Caroline DESMETZ |
| E-mail(s) |
[email protected]
|
| Organization name |
Montpellier University
|
| Department |
Faculty of pharmacy
|
| Lab |
EA7288-BC2M
|
| Street address |
15 avenue Charles Flahault
|
| City |
Montpellier |
| ZIP/Postal code |
34093 |
| Country |
France |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE84524 |
Effect of a salt restriction diet on cardiac damage in a rat model of metabolic syndrome |
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