|
| Status |
Public on Sep 30, 2024 |
| Title |
Whole_blood_LRFI_30_4420_2 |
| Sample type |
RNA |
| |
|
| Source name |
Whole blood
|
| Organism |
Sus scrofa |
| Characteristics |
microarray_number: 11
microarray_position: 7
repetition: 2
pig_number: 4420
breed: LRFI
pen: 17
day_of_experiment: 2
day_hour: 2
temperature: 30
pig_day_hour: 4420_2
gender: neuter
|
| Treatment protocol |
Whole blood RNA were collected on PAXgene Blood RNA Tubes (PreAnalytiX) and were conserved at -20°C until extraction.
|
| Growth protocol |
The pigs were raised in selection facilities (INRAE, GenESI, Le Magneraud, France, doi : 10.15454/1.5572415481185847E12), then transferred to the experimental facilities of INRAE in Saint-Gilles (Pig Physiology and Phenotyping Experimental Facilit, doi 10.15454/1.5573932732039927E12) at approximately 80 days of age (i.e., 40 kg of BW).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted according to the manufacturer's recommendations (PAXgene Blood RNA Kit, Qiagen) and the extracted total RNA was eluted in 40 µl of buffer BR5 and stored at -80°C. Quantification was performed using a Nanodrop to determine the concentration of each purification and the 260/230, 260/280 ratios. Quality was checked by electrophoresis on 0,8% agarose gel. RNA Integrity Numbers (RINs) were determined using a 2100 Bioanalyzer Instrument (Agilent).
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 100 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).
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8 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 |
castrated
|
| 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. Probes were filtered and kept if they had a good signal in more than 75% of the samples in at least one experimental group. Using this filter, 32924 probes were selected from a total of 61625, or 53.4%, normalized by the median, and log2 transformed. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. A chip as batch effect was corrected with using the ComBat procedure from the sva package. The resulting matrix has 32924 rows each corresponding to a unique ProbeName (provided as data Matrix) and 75 columns (pigs x day +/- hour).
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| |
|
| Submission date |
Jul 02, 2024 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Laurence Liaubet |
| E-mail(s) |
laurence.liaubet@inrae.fr
|
| Phone |
+33 561285113
|
| Organization name |
INRAE
|
| Department |
Animal genetics
|
| Lab |
GenPhySE
|
| Street address |
24 chemin de Borde Rouge
|
| City |
Castanet-Tolosan |
| ZIP/Postal code |
31326 |
| Country |
France |
| |
|
| Platform ID |
GPL19893 |
| Series (1) |
| GSE271309 |
Kinetics of the whole blood transcriptome in response to heat stress in pigs from two lines divergently selected for feed efficiency. |
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