|
| Status |
Public on Mar 24, 2018 |
| Title |
Vitamine D depletion followed by repletion diet, biological replicate #4 |
| Sample type |
RNA |
| |
|
| Source name |
plantaris skeletal muscle
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar
diet: depletion and repletion of vitamin D in the diet
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol (Life Technologies) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Low Input Quick Amp labeling One color : Cy3 (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Rat GE 4*44K V3 Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters . Data treatment was performed with bioconductor and Limma library. Background substraction was performed using the Normexp correction with offset =50. Quantile normalization was then applied. Features flagged in Feature Extraction as control, as Feature Non-uniform outliers, saturating or too weak were excluded.
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| |
|
| Submission date |
Mar 25, 2015 |
| Last update date |
Mar 24, 2018 |
| Contact name |
emmanuelle meugnier |
| E-mail(s) |
[email protected]
|
| Organization name |
INSERMU1060/INRAE1397
|
| Lab |
CarMeN
|
| Street address |
Cens Eli Bat 2 D- Hopital Lyon Sud
|
| City |
Pierre Bénite |
| ZIP/Postal code |
69495 |
| Country |
France |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE67274 |
Impact of Vitamine D depletion and repletion on rat skeletal muscle |
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