|
| Status |
Public on Sep 20, 2008 |
| Title |
male chicken BC2 n°4114 (from the sire: BC1 n°1940) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
male chicken BC2 n°4114 (from the sire: BC1 n°1940)
|
| Organism |
Gallus gallus |
| Characteristics |
chicken type: broiler
gender: male
age: 9 weeks
tissue: liver
|
| Growth protocol |
Standard broiler chicken growth protocol. Livers were rapidly excised, frozen in liquid nitrogen and stored at –75 °C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA)
|
| Label |
Cy5
|
| Label protocol |
Five µg of each mRNA sample were reverse-transcribed and Cy5 fluorescent-labeled using the ChipShot™ Direct Labeling kit (Promega, Charbonnieres, France).
|
| |
|
| Channel 2 |
| Source name |
Reference
|
| Organism |
Gallus gallus |
| Characteristics |
chicken type: broiler
gender: male
age: 9 weeks
tissue: liver
sample type: total liver RNA pool obtained from the 48 male broiler chickens (sire BC1 n°1940)
|
| Growth protocol |
Standard broiler chicken growth protocol. Livers were rapidly excised, frozen in liquid nitrogen and stored at –75 °C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver using TRIzol reagent according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA)
|
| Label |
Cy3
|
| Label protocol |
Five µg of each mRNA sample were reverse-transcribed and Cy3 fluorescent-labeled using the ChipShot™ Direct Labeling kit (Promega, Charbonnieres, France).
|
| |
|
| |
| Hybridization protocol |
The microarray was hybridized for 8 h at 42°C with Cy5-labeled chicken cDNA sample and an aliquot of the Cy3-labeled reference cDNA using automatic hybridization chamber (Discovery from Ventana Medical System, Inc). Slides were washed in increasing stringency using salt sodium citrate.
|
| Scan protocol |
The microarray slides were scanned using a laser scanner (GenePix 4000A from Axon Instrument, CA) at 550 nM for Cy3 and 640 nM for Cy5 to generate two TIFF images for each slide.
|
| Description |
Male broiler chicken fed ad libitum during 4 hours minimum.
For each gene, the fluorescence ratio represents the relative abundance of the hepatic mRNA of the animal of interest compared with the RNA pool reference.
|
| Data processing |
The two images from each slide were analyzed with GenepixPro 4.0 software (Axon instruments, Inc., Union City, CA). Pre-processing and normalization of data were accomplished using the R-project statistical and Bioconductor environment (http://www.r-project.org) (http://www.bioconductor.org).
Data filtering: the first step was to select the genes considered as expressed in liver (roughly 60% expected) regarding to a good contrast between spot and background intensities (SNR (Signal to Noise Ratio) defined superior to 2). Among the 20461 genes on microarray, we selected 11590 genes (57%) and eliminated one microarray (n°5643) for which the average SNR was equal to 1.5. Second, we removed 377 genes for which minimum of 20% of spots among the 45 microarrays did not respect the 2 criteria: i) absence of the genepix flag (automatically performed by GenepixPro 4.0) and ii) a symmetry of the spot. A total of 11213 genes were finally selected. Third we targeted the isolated “bad” spots not conformed to the two criteria in order not to take into account during the normalization procedure: 2.8% of spots were concerned.
Data normalization: the Cy5/Cy3 ratio used was expressed as the Log2 of the ratio of median pixel intensity of the two red and green spots. Median Log2 ratio values were then normalized (ratio centered on zero) by Lowess fitness. Only the array spots conforming to the filtering step were used for the normalization.
|
| |
|
| Submission date |
Aug 02, 2008 |
| Last update date |
Jun 04, 2010 |
| Contact name |
Sandrine Lagarrigue |
| E-mail(s) |
sandrine.lagarrigue@agrocampus-rennes.fr
|
| Phone |
33 02 23 48 59 59
|
| Fax |
33 02 23 48 54 70
|
| Organization name |
Agrocampus Rennes/ INRA
|
| Lab |
UMR Genetique animale
|
| Street address |
65 rue de St Brieuc
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL5480 |
| Series (1) |
| GSE12319 |
Chicken family BC1 1940: transcriptome profiling used for QTL/eQTL detection |
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