|
| Status |
Public on Jun 25, 2013 |
| Title |
28d p.i. Salmonella |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S.typhimurium infected
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: ileum
infection: S.typhimurium
treatment: no treatment
time: 28d
|
| Treatment protocol |
piglets in S and SP group were infected with S.typhimurium DT104 at day 28 postpartum directly after weaning; piglets in SP group had free access to feed supplemented with the probiotic strain E.faecium NCIMB 10415
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ntestinal samples (~ 2 cm circle segments) were taken at time points 3h, 3d and 28d post infection from ileum of ten S.typhimurium infected piglets (S), ten S.typhimurium infected piglets and additional treated with E.faecium (SP) and five healthy control piglets(C) (EUROC x Pietrain). The piglets were weaned at the age of 28 days and infected with S. typhimurium. Samples were quick-frozen in liquid nitrogen and stored at -80 °C. In order to obtain representative measurements in each intestinal locus, three cross sections of approximately 2 mm out of the 2 cm segment of frozen intestine were examined. These 3 sections were pooled and total RNA was isolated from samples using an automated homogenizer (FastPrep Instrument, MP Biomedicals, Heidelberg, Germany) and the mirVana miRNA Isolation Kit (Applied Biosystems, Darmstadt, Germany), according to the manufacturer’s protocol. The RNA quality and quantity of all samples were proven using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kits (Agilent, Waldbronn, Germany) and the Nanodrop 1000 Spectrophotometer (Thermo Scientific, MA, USA).
|
| Label |
Cy5
|
| Label protocol |
Total RNA was converted into cDNA and fluorescently labelled using the SuperScript Plus Indirect cDNA Labelling System (Life technologies) and the Alexa Fluor Dyes according to the manufacturor's protocol.
|
| |
|
| Channel 2 |
| Source name |
common reference
|
| Organism |
Sus scrofa |
| Characteristics |
composition: equal amounts of total RNA of each ileum pool and additional total RNA from ascending colon
|
| Treatment protocol |
piglets in S and SP group were infected with S.typhimurium DT104 at day 28 postpartum directly after weaning; piglets in SP group had free access to feed supplemented with the probiotic strain E.faecium NCIMB 10415
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ntestinal samples (~ 2 cm circle segments) were taken at time points 3h, 3d and 28d post infection from ileum of ten S.typhimurium infected piglets (S), ten S.typhimurium infected piglets and additional treated with E.faecium (SP) and five healthy control piglets(C) (EUROC x Pietrain). The piglets were weaned at the age of 28 days and infected with S. typhimurium. Samples were quick-frozen in liquid nitrogen and stored at -80 °C. In order to obtain representative measurements in each intestinal locus, three cross sections of approximately 2 mm out of the 2 cm segment of frozen intestine were examined. These 3 sections were pooled and total RNA was isolated from samples using an automated homogenizer (FastPrep Instrument, MP Biomedicals, Heidelberg, Germany) and the mirVana miRNA Isolation Kit (Applied Biosystems, Darmstadt, Germany), according to the manufacturer’s protocol. The RNA quality and quantity of all samples were proven using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kits (Agilent, Waldbronn, Germany) and the Nanodrop 1000 Spectrophotometer (Thermo Scientific, MA, USA).
|
| Label |
Cy3
|
| Label protocol |
Total RNA was converted into cDNA and fluorescently labelled using the SuperScript Plus Indirect cDNA Labelling System (Life technologies) and the Alexa Fluor Dyes according to the manufacturor's protocol.
|
| |
|
| |
| Hybridization protocol |
Fully automated hybridisation and washings were carried out in the aHyb™ Hybridization Station (Miltenyi Biotec). Firts, slides were pre-hybridised at 45°C for 5 min in pre-hybridisation buffer (5xSSC, 0.1%SDS, 1%BSA). Hybridisation was performed for 16h at 50°C with a pump rate of 1 ml/min. Finally the slides were washed in wash buffers with increased stringency. At first for 1 min at 50°C in wash buffer 1 (2xSSC, 0.5%SDS), followed by wash buffer 2 (0.5$SSC, 0.5%SDS), and wash buffer 3 (0.1%SSC, 0.2%SDS) each 1 min at 25°C. Afterwards, dipped in bi-distilled water dried by brief centrifugation.
|
| Scan protocol |
The microarrays were scanned at 10 µm resolution using a GenePix 4000B scanner (Molecular Devices).
|
| Description |
28d p.i. S [mRNA]
|
| Data processing |
Microarray image analysis and ratio-based normalisation of the microarray data was conducted using the software package GenePix Pro 6.1 (Molecular Devices). Data was filtered according following criteria: [SNR 635] < 3 AND [SNR 532] < 3 AND [% > B532 + 2 SD] < 70 OR [% > B 635 + 2 SD] < 70 OR [F 532 Median – B 532] < 500 AND [F 635 Median – B 635] < 500.
|
| |
|
| Submission date |
Sep 12, 2012 |
| Last update date |
Jun 25, 2013 |
| Contact name |
Soroush Sharbati |
| E-mail(s) |
sharbati@zedat.fu-berlin.de
|
| Organization name |
Freie Universitaet Berlin
|
| Department |
Institute of Veterinary Biochemistry
|
| Street address |
Oertzenweg 19b
|
| City |
Berlin |
| ZIP/Postal code |
14163 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16045 |
| Series (2) |
| GSE40804 |
Intestinal Salmonella Typhimurium infection leads to miR-29a induced Caveolin 2 regulation [mRNA] |
| GSE40805 |
Intestinal Salmonella Typhimurium infection leads to miR-29a induced Caveolin 2 regulation |
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