|
| Status |
Public on Nov 23, 2017 |
| Title |
03PBMC_HF_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
PBMC_HF_rep1
|
| Organism |
Gallus gallus |
| Characteristics |
tissue: Peripheral Blood Mononuclear Cells
gender: male
diet: HF
|
| Treatment protocol |
At 63 days of age, the liver, Pectoralis major muscle and abdominal fat were immediately removed and frozen in liquid nitrogen and then stored at -80°C until analyses. Peripheral blood mononucleated cells (PBMC) were isolated, just after sampling by density gradient centrifugation (Ficoll Histopaque).
|
| Growth protocol |
2 x 12 chickens per line were placed in individual cages and fed two different diets from 21 to 63 d. These two diets were isocaloric (12.54 MJ ME/kg) and isonitrogenous (190 g CP/kg) but exhibited a high-starch and low-fiber low-lipid contents (LF diet) or a low-starch and high-fiber high-lipid contents (HF diet).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol®. The quantity and quality of extracted RNA was evaluated using the NanoDrop ND-1000 spectrophotometer and the Agilent 2100 BioAnalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 150ng RNA using the Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent custom 8 x 60K chicken gene expression 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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the the Agilent DNA Mmicroarray scanner using one color scan setting for 8x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (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.
|
| |
|
| Submission date |
Sep 20, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Colette DESERT |
| E-mail(s) |
[email protected]
|
| Organization name |
Agrocampus INRA UMR1348 PEGASE
|
| Street address |
65 Rue de St Brieuc
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL19630 |
| Series (2) |
| GSE104041 |
Transcriptome profile of liver, adipose tissue, muscle and PBMC in genetically fat and lean chicken lines submitted to high-fiber and high-fat diet versus standard high-starch diet [PBMC] |
| GSE104042 |
Transcriptome profile of liver, adipose tissue, muscle and PBMC in genetically fat and lean chicken lines submitted to high-fiber and high-fat diet versus standard high-starch diet |
|
