|
| Status |
Public on Sep 19, 2014 |
| Title |
Fetus686_d110_LWLW |
| Sample type |
RNA |
| |
|
| Source name |
skeletal muscle Longissimus lumborum (LD)
|
| Organism |
Sus scrofa |
| Characteristics |
day of gestation: 110
fetus genotype: LWLW
maternal genotype: LW
paternal genotype: LW
|
| Treatment protocol |
Muscle samples were disrupted, homogenized and ground to a fine powder by rapid agitation for 1 minute in a liquid-nitrogen cooled grinder with stainless steel beads. RNA samples were conserved at -80°C until extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An aliquot of 100mg of the fine powder was then processed for total RNA isolation and purification using Trizol (Invitrogen, France) and the kit Nucleospin RNA II (Macherey-Nagel, France) according to the manufacturer's instructions. The method included a DNase digestion step to remove contaminating DNA. The extracted total RNA was eluted in 300µl of RNase-free water and stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy purification (QIAGEN). Dye incorporation and cRNA yield were checked with the Biospec nano spectrophotometer (Shimadzu).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse GE microarray (8X60K, Design 028005) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min), Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
|
| Scan protocol |
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software.
|
| Description |
Gene expression at 110 days of gestation in LWLW fetal skeletal muscle
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 037880_D_F_2012). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 44368 rows each corresponding to a unique ProbeName (provided as data Matrix) and 61 columns (individuals).
|
| |
|
| Submission date |
Mar 27, 2014 |
| Last update date |
Sep 20, 2014 |
| Contact name |
Laurence Liaubet |
| E-mail(s) |
laurence.liaubet@inra.fr
|
| Organization name |
INRA
|
| Department |
Animal Genetic
|
| Lab |
GenPhySE
|
| Street address |
chemin de borde rouge
|
| City |
Castanet tolosan |
| ZIP/Postal code |
31326 |
| Country |
France |
| |
|
| Platform ID |
GPL16524 |
| Series (1) |
| GSE56301 |
Muscle Transcriptomic Investigation of Late Fetal Development Identifies Candidate Genes for Key Roles in Piglet Maturity |
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