|
| Status |
Public on May 05, 2010 |
| Title |
Mono cultures 12 h rep1 _ Mono cultures 8 h rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mono cultures grown for 12 h (RNA isolated from S. thermophilus and L. bulgaricus mono cultures was mixed)
|
| Organisms |
Streptococcus thermophilus CNRZ1066; Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 |
| Characteristics |
strain: Streptococcus thermophilus CNRZ1066
strain: Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365
|
| Treatment protocol |
Yoghurt cultures were quenched in 3 volumes 60% glycerol of -40°C and kept at -20°C for 0.5 h. The pH was adjusted to 6.5 – 7.0 with 1 M NaOH. The medium was cleared by adding 4 mL 25% (w/v) trisodium citrate per 100 mL. The sample was gently mixed each 5 min for 0.5 h while keeping it at -20°C. Cells were centrifuged at -20°C and 23,000 G for 16 min. In order to remove residual protein, the cell pellet was resuspended in a solution comprised of 50% (w/v) guanidinethiocyanate (Sigma), 0.5% (w/v) N-laurylsarcosine (Sigma) and 2.5% (v/v) of a 1 M trisodium citrate solution, adjusted to pH 7.0. After centrifugation, the cells were resuspended in 500 µL 1xTE (10xTE contains 10 mM tris pH 8.0 and 1 mM EDTA pH 8.0) and transferred to a screw-cap tube.
|
| Growth protocol |
Cells were inoculated at an OD600 of 0.005 and grown in 275 mL reconstituted skim milk at 42°C for 3.5, 5.5, 8 or 12 h, dependent on the sampling time.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a phenol chloroform extraction followed by column-purification. Cells were disrupted by shaking 3 times 45 s in a Fastprep (Qbiogene Inc., France) at 5.5 m/s separated by 1 min on ice.
|
| Label |
Cy3
|
| Label protocol |
Five to seven μg of RNA was used for cDNA synthesis and labelling using the Cyscribe Post-labelling kit (Amersham Biosciences, Amersham, UK) according to the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Mono cultures grown for 8 h (RNA isolated from S. thermophilus and L. bulgaricus mono cultures was mixed)
|
| Organisms |
Streptococcus thermophilus CNRZ1066; Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 |
| Characteristics |
strain: Streptococcus thermophilus CNRZ1066
strain: Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365
|
| Treatment protocol |
Yoghurt cultures were quenched in 3 volumes 60% glycerol of -40°C and kept at -20°C for 0.5 h. The pH was adjusted to 6.5 – 7.0 with 1 M NaOH. The medium was cleared by adding 4 mL 25% (w/v) trisodium citrate per 100 mL. The sample was gently mixed each 5 min for 0.5 h while keeping it at -20°C. Cells were centrifuged at -20°C and 23,000 G for 16 min. In order to remove residual protein, the cell pellet was resuspended in a solution comprised of 50% (w/v) guanidinethiocyanate (Sigma), 0.5% (w/v) N-laurylsarcosine (Sigma) and 2.5% (v/v) of a 1 M trisodium citrate solution, adjusted to pH 7.0. After centrifugation, the cells were resuspended in 500 µL 1xTE (10xTE contains 10 mM tris pH 8.0 and 1 mM EDTA pH 8.0) and transferred to a screw-cap tube.
|
| Growth protocol |
Cells were inoculated at an OD600 of 0.005 and grown in 275 mL reconstituted skim milk at 42°C for 3.5, 5.5, 8 or 12 h, dependent on the sampling time.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a phenol chloroform extraction followed by column-purification. Cells were disrupted by shaking 3 times 45 s in a Fastprep (Qbiogene Inc., France) at 5.5 m/s separated by 1 min on ice.
|
| Label |
Cy5
|
| Label protocol |
Five to seven μg of RNA was used for cDNA synthesis and labelling using the Cyscribe Post-labelling kit (Amersham Biosciences, Amersham, UK) according to the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
For each array, 0.3 µg of cDNA labeled with either Cyanine 3 or Cyanine 5 was hybridized using the solutions and following the protocol delivered by Agilent (version 5.5) for 8x15K slides.
|
| Scan protocol |
Slides were scanned using an Agilent microarray scanner (G2565BA). Lasers of wavelengths at 532 and 635 nm were used to excite Cyanine3 and Cyanine5 dyes, respectively. Fluorescent images were captured as multi-image-tagged image file format and analyzed with Imagene software (Axon) (BioDiscovery, Marina del Rey, USA).
|
| Description |
Mono cultures in milk grown for 12 h rep1 compared to Mono cultures in milk grown for 8 h rep1
|
| Data processing |
The extent of hybridization was derived from a median value of pixel-by-pixel ratios. S. thermophilus and L. bulgaricus spots were normalized using Lowess separately as a varying ratio between both organisms would impact the determined expression of genes in each species. Differential regulation was determined by false-discovery rate (FDR) from the Cyber-T p-values by means of multiple testing connections. Differential regulation was defined as a two-fold or higher differential expression with a FDR cut-off value of 0.05 or lower.
|
| |
|
| Submission date |
Apr 29, 2010 |
| Last update date |
May 04, 2010 |
| Contact name |
Sander Sieuwerts |
| Organization name |
NIZO Food Research
|
| Department |
Health
|
| Street address |
Kernhemseweg 2
|
| City |
Ede |
| ZIP/Postal code |
6718ZB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10366 |
| Series (1) |
| GSE21593 |
Interactions between Streptococcus thermophilus and Lactobacillus bulgaricus in yoghurt |
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