|
| Status |
Public on Sep 21, 2011 |
| Title |
caecum_germ_free_non_inoculated_(pool) |
| Sample type |
RNA |
| |
|
| Source name |
caecum from germ-free NMRI-KI mice, not inoculated, pooled samples
|
| Organism |
Mus musculus |
| Characteristics |
strain: NMRI-KI
gender: female
age: 45-60 days
developmental stage: adult
tissue: caecum
bacterial inoculation: none
|
| Treatment protocol |
The germ-free female NMRI-KI mice (45 – 65 days of age) were mono-associated using established protocols. In brief, 10 ml of cultures in late log phase of A. muciniphila MucT (ATTC BAA-835) and L. plantarum WCFS1 (NCIMB 8826) were centrifuged (4500 rpm, 10 min). Pellets were resuspended in 1 ml of sterile anaerobic Phosphate Buffer Saline (PBS) and dispensed into sterile ampoules which were heat-sealed. The external surface of each ampoule was sterilized with chromsulfuric acid before transfer into respective isolators. Inside the isolators, the ampoules were broken, and 0.2 ml (10^9 cfu/ml) of the strictly anaerobic A. muciniphila (n=6) was inoculated intragastrically; L. plantarum (n=6) was inoculated orally. Germ-free control mice (n=6) were housed in separate isolators. After 7 days of colonization, mice were killed by cervical dislocation and terminal ileum, cecum and ascending colon specimens were sampled.
|
| Growth protocol |
Germ-free female NMRI-KI mice were inbred for >60 generations at the Laboratory of Medical Microbial Ecology at Karolinska Institute and they were housed in lightweight stainless-steel isolators. All mice had free access to a steam-sterilized standard mouse chow (R36; Lactamin, Vadstena, Sweden) and to sterilized water. Artificial light was available between 6 a.m. and 6 p.m.; the temperature was 24 ± 2.2°C, and the humidity was 55% ± 10%. The germ-free status was checked weekly by inoculating fecal samples in different media incubated both aerobically and anaerobically at 20 and 37°C for up to 4 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After sampling, tissues were immediately preserved in 5 volumes of RNAlater (Ambion, Austin, Texas, USA) and stored at 4 ºC until use. Total RNA was prepared using TRIzol reagent and whereafter Purified total RNA was isolated using Qiagen RNEasy columns. For each part of the intestine, total RNA was pooled per intervention group (n=6).
|
| Label |
biotin
|
| Label protocol |
The Affymetrix GeneChip RNA One cycle Amplification Kit was used to prepare labelled cRNA from 5 ug of total RNA. The protocol was conducted using the reagents provided by Affymetrix in the One-Cycle cDNA synthesis kit (P/N 900431), One-Cycle IVT labelling kit (P/N 90449) and GeneChip Sample Cleanup Module (P/N 900371). A detailed description can be found in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 1 (Eukaryotic Target Preparation) (P/N 701025, revision 6).
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| |
|
| Hybridization protocol |
Hybridisation of 10ug cRNA was done overnight for 16 hours at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol is conducted as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Target Hybridization) (P/N 701027, revision 5).
|
| Scan protocol |
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
|
| Description |
A54_04_CAECUM_NEG CTRL
|
| Data processing |
Expression estimates were calculated using the multi-chip modified gamma Model for Oligonucleotide Signal (multi-mgMOS) [PMID: 16020470], using the PUMA package in Bioconductor.
|
| |
|
| Submission date |
Oct 15, 2009 |
| Last update date |
Sep 21, 2011 |
| Contact name |
Guido Hooiveld |
| E-mail(s) |
guido.hooiveld@wur.nl
|
| Organization name |
Wageningen University
|
| Department |
Div. Human Nutrition & Health
|
| Lab |
Nutrition, Metabolism & Genomics Group
|
| Street address |
HELIX, Stippeneng 4
|
| City |
Wageningen |
| ZIP/Postal code |
NL-6708WE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE18587 |
Specific modulation of mucosal immune response, tolerance and proliferation in mice colonized with A. muciniphila |
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