|
| Status |
Public on Nov 24, 2015 |
| Title |
LWF(M_2_05) |
| Sample type |
RNA |
| |
|
| Source name |
skeletal muscle Longissimus lumborum (LD)
|
| Organism |
Sus scrofa |
| Characteristics |
breed: Large White Femelle
|
| Treatment protocol |
Muscle samples were biopsied from Longissimus lumborum (LD) muscle 20’ after stunning and exsanguination. Samples were immediately frozen in liquid N2 and kept at -80°C until analysis.
|
| Growth protocol |
The animals were raised at the French central test Station in Le Rheu (France) in 2007 and 2008.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The muscle samples were disrupted, homogenized and ground to a fine powder by rapid agitation for 1 min in a liquid-nitrogen cooled grinder with stainless steel beads. An aliquot of 250–300 mg of the fine powder was then processed for total RNA isolation and purification using RNeasy Fibrous Tissue Midi kit according to the manufacturer’s instructions (Qiagen SA France, Courtaboeuf, France). The method included a proteinase K digestion step to remove proteins and a DNase digestion step to remove contaminating DNA. The extracted total RNA was eluted in 300 µl of RNase-free water and stored at -80°C. RNA quality and concentration were controlled using an AGILENT 2100 bioanalyzer (RNA solutions and RNA 6000 Nano Lab- Chip Kit, Agilent Technologies France, Massy, France)
|
| 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 (4X44K) 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 scanner 4000B de Axon with Feature Extraction V 9.5.3.1 software
|
| Data processing |
The scanned images were analyzed with Feature Extraction V 9.5.3.1 (Agilent) . 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. The whole set of 45,220 spots were analysed. Each spot on each chip was allocated a 0-1 weight depending on various criteria on quality (routine pipeline of the Plateforme TRIX). On average, half of the spots per chip were dropped. Only spots with good quality (weight 1) for at least 70% of the chips were kept for further analysis, leading to 12,358 spots. All intensity data were log transformed, and the term “data” or “intensity” will refer to log transformed data from now on (natural logarithm). The effect of the hybridization day was removed by subtracting the mean intensity of the hybridization day. Then the mean of each chip was subtracted to make all chips comparable. A good linearity of intensity signals were observed between all samples. The resulting matrix has 12358 rows each corresponding to a unique ID (provided as data Matrix) and 147 columns (individuals).
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| |
|
| Submission date |
Mar 18, 2014 |
| Last update date |
Nov 24, 2015 |
| 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 |
GPL18437 |
| Series (1) |
| GSE56011 |
Exploring transcriptomic diversity in muscle revealed that cellular signaling pathways mainly differentiate five Western porcine breeds |
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