|
| Status |
Public on Jan 31, 2016 |
| Title |
skin antigen (animal n°3 sample7) |
| Sample type |
RNA |
| |
|
| Source name |
skin
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: skin
treatment: 15µg H1 N1 + PBS buffer
line: TOPIG 20
Sex: male
age: 9 weeks-old
|
| Treatment protocol |
Pigs (Sus scrofa domestica, line TOPIG 20, 9 week old male animals, Lelystad) were first tested free of influenza antibodies using influenza hemagglutination inhibition assay. Animals were then anesthetized with Propofol and were immunized at various sites of the skin with various formulations (a total of 7 sites per animal). The formulations of vaccines were all done in phosphate buffered saline [PBS]. The TLR5 agonist: the flagellin FliC from Salmonella enterica serovar Typhimurium was administered at various doses (0.1 to 10 micrograms) in combination with 15 micrograms of HA antigen from H1N1 influenza virus (cGMP grade) diluted in 100 microliters of PBS. Control sites were also injected with PBS alone or HA antigen from H1N1 in PBS. The intradermal administration was done using 29G x 1/2 hypodermic needle. The skin area of injection was marked for later sampling the site of flagellin administration. Finally, mock skin, i.e. un-injected skin was used as an additional negative control for microarrays.
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Animals were anesthetized at 4h post-immunization using Ketamin/Dormicum (4 animals per formulations/controls). Punch biopsies of skin were sampled using 8 mm diameter biopsy punch and frozen in liquid nitrogen. Total RNA from skin was extracted using TRI Reagent® (Ambion) method according to supplier's instructions after homogenization using Ultra Turrax homogenizer. Finally, total RNA was extracted again by column-based method using Nucleospin II RNA kit (Macherey-Nagel).
|
| Label |
Cy3
|
| Label protocol |
Total RNA labeling was performed with the single color Quick Amp Labeling Kit (Agilent Technologies).
|
| |
|
| Hybridization protocol |
After precipitation, purification and quantification, 1.25µg labeled samples were hybridized according to the supplier’s protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 5µm resolution and extended range using a G2565CA high resolution laser microarray scanner (Agilent Technologies).
|
| Data processing |
Microarray image data were extracted and analyzed with the Image Analysis / Feature Extraction software G2567AA (Version A.10.10.1, Agilent Technologies). The extracted MAGE-ML files were further analyzed with the Rosetta Resolver, Build 7.2.2.0 SP1.31 (Rosetta Biosoftware, Seattle, USA).
|
| |
|
| Submission date |
Aug 20, 2015 |
| Last update date |
Aug 28, 2016 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15007 |
| Series (1) |
| GSE72256 |
Pig-specific skin transcriptional responses after intradermal administration of flagellin |
|
