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| Status |
Public on Jul 01, 2011 |
| Title |
B. breve UCC2003 grown in MRS vs B. breve UCC2003 grown in vivo experiment 3 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Bifidobacterium breve UCC2003 grown in MRS
|
| Organism |
Bifidobacterium breve UCC2003 |
| Characteristics |
strain: UCC2003
growth stage: exponential
od600: 0.5
carbon source: glucose
|
| Biomaterial provider |
Douwe van Sinderen, Department of Microbiology, University College Cork, Ireland
|
| Treatment protocol |
Method for cell disruption was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
|
| Growth protocol |
Bifidobacterium breve UCC200 was grown in modified de Man, Rogosa and Sharpe (mMRS) medium (De Man, J. C., A. Rogosa & M. E. Sharpe, (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135), made from first principles, supplemented with 0.05% (w/v) L-cysteine-HCl (Sigma-Aldrich, Steinhein, Germany) and 1% (w/v) of glucose solution as the sole carbon source. Strain was grown under anaerobic conditions in a Modular Atmosphere Controlled System (Davidson & Hardy Ltd., Dublin, Ireland) till OD600 was 0.5 .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation, RNA quality control was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
|
| Label |
Cy3
|
| Label protocol |
cDNA from total RNA was synthesized using the cDNA synthesis and labelling kit DSK-001 (Kreatech) according to the manufacturer’s instructions
1 μl of each cDNA solution was labelled with Cy3 using Cy3-ULS from the cDNA synthesis and labelling kit DSK-001 (Kreatech, Amsterdam, The Netherlands) according to the manufacterers instructions.
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| Channel 2 |
| Source name |
ceaca of Balb/C mice colonized with B. breve UCC2003
|
| Organism |
Mus musculus |
| Characteristics |
strain: MUCC2003
source tissue: Mus musculus balb/c ceacum
age: 25 days
|
| Biomaterial provider |
Douwe van Sinderen, Department of Microbiology, University College Cork, Ireland
|
| Treatment protocol |
Caeca, isolated from mice, were placed in RNAlater (Ambion) and immediately processed. Method for cell disruption was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77)
|
| Growth protocol |
Seven-week-old male, BalbC mice were housed in individually vented cages (Animal care systems, Colorado) under a strict 12 h light cycle. Mice (n = 5 per group) were fed a standard polysaccharide-rich mouse chow diet and water ad libidum. Mice were inoculated by oral gavage (109 cfu of B. breve UCC2003 pPKCM7 in 100 ul of PBS ). Fecal pellets were collected at intervals over 25 days to enumerate bacteria. Twenty five days after inoculation, mice were sacrificed and their intestinal tracts quickly dissected. Aliquots of small intestine, caecum and large intestine were harvested for determination of colony forming units (cfu) (serial dilution plating on RCA agar plates with appropriate antibiotics). Caeca were placed in RNAlater for immediate RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial mRNA was extracted from caecal RNA preparations using the MicrobEnrich and MicrobExpress kits (Ambion) according to the manufacturer’s instructions. cDNA from bacterial mRNA was synthesized using the cDNA synthesis and labelling kit DSK-001 (Kreatech) according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
1 μl of each cDNA solution (10 ng) was amplified in triplicate using the GenomiPhi™ V2 DNA Amplification Kit (Amersham Biosciences) according to the manufacturer's protocol and labelled with Cy5 using Cy5-ULS from the cDNA synthesis and labelling kit DSK-001 (Kreatech).
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| Hybridization protocol |
Labelled and amplified cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis (v4.0) manual (publication number G4140-90050).
|
| Scan protocol |
Following hybridization, microarrays were washed as described in the manuals and scanned using Agilent's DNA microarray scanner G2565A.
|
| Data processing |
The scanning results were converted to data files with Agilent's Feature Extraction software (version 9.5) using standard settings as described in the Agilent's Feature Extraction manual.
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| Submission date |
Feb 24, 2011 |
| Last update date |
Jul 01, 2011 |
| Contact name |
Aldert Zomer |
| E-mail(s) |
A.L.Zomer@uu.nl
|
| Organization name |
Utrecht University
|
| Department |
Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine
|
| Street address |
Yalelaan 1
|
| City |
Utrecht |
| ZIP/Postal code |
3584 cl |
| Country |
Netherlands |
| |
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| Platform ID |
GPL13210 |
| Series (1) |
| GSE27491 |
Functional genome analysis of Bifidobacterium breve UCC2003 reveals a major conserved host-colonization factor |
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