|
| Status |
Public on Oct 10, 2012 |
| Title |
TC01 bile_3 |
| Sample type |
RNA |
| |
|
| Source name |
Cultured cells
|
| Organism |
Lacticaseibacillus casei |
| Characteristics |
agent: bile
strain: TC01. L. casei BL23 with a complete deletion of gene LCABL_02080.
|
| Treatment protocol |
Cells were grown in MRS medium at 37C without shaking until O.D. 595 nm. reached 0.5. The culture was then two fold diluted with prewarmed MRS medium supplemented with 0.2% bovine bile (Sigma) and incubation continued for 45 min.
|
| Growth protocol |
Cells were stored at -80C in MRS medium supplemented with 15% glycerol (stock culture). For the assay, cells from the stock culture were inoculated in MRS agar plates. A single colony was used to inoculate 5 ml of MRS medium and the culture was incubated overnight at 37C. This culture was used to inoculate MRS medium at 0.05 O.D. 595 nm. for the treatment or control assays.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from L. casei by using TRIZOL reagent (Invitrogen). Cells were collected by centrifugation, washed with cold 50 mM EDTA (pH 8.0), resuspended in 1 ml of TRIZOL reagent, and then mechanically disrupted with glass beads in a cell disrupter (Savant Instruments, Holbrook, N.Y.). Additional steps were performed according to the instructions of the TRIZOL manufacturer. RNA samples purified as described above were treated with the Ambion Turbo DNA-free kit (Applied Biosystems) using the routine DNase I treatment outlined by the supplier in order to remove contaminating DNA. The quality and concentration of the RNA samples were subsequently evaluated by using the Experion automated electrophoresis system (Bio-Rad). Samples with 23S/16S ratios lower than 0.85 were discarded.
|
| Label |
33P-dCTP
|
| Label protocol |
First-strand cDNA was synthesized from 45 µg RNA using the Transcriptor first strand cdna synthesis kit (Roche) with random hexamer primers as recommended by the manufacturer. Labelled cDNA was purified with illustra MicroSpin G-50 Columns (GE Healthcare) as recommended by the manufacturer.
|
| |
|
| Hybridization protocol |
SDS based hybridization solution, during 24 hours
|
| Scan protocol |
FUJIFILM FLA-3000
|
| Data processing |
The software for image analysis is ArrayVision 7.0 (Imaging Research Inc.). This software yields raw and background substracted data.
The Background Substracted Signals are raw individual data. After considering the three replicates performed in the experiment and after median normalization, first between replicates and after between conditions, we obtain the ratio values for each gene/ORF (see supplementary file on Series record).
|
| |
|
| Submission date |
Nov 17, 2010 |
| Last update date |
Oct 10, 2012 |
| Contact name |
Manuel Zúñiga |
| E-mail(s) |
btcman@iata.csic.es
|
| Phone |
34 963900022
|
| Fax |
34 963636301
|
| Organization name |
Instituto de Agroquímica y Tecnología de Alimentos
|
| Department |
Biotecnología
|
| Lab |
Bacterias lácticas y Probióticos
|
| Street address |
Avda. Profesor Agustín Escardino 7
|
| City |
Paterna |
| State/province |
Valencia |
| ZIP/Postal code |
46980 |
| Country |
Spain |
| |
|
| Platform ID |
GPL11074 |
| Series (1) |
| GSE25448 |
Comparison of the response to bile of Lactobacillus casei BL23 and its derivative strain TC01 |
|
