|
| Status |
Public on Jan 17, 2008 |
| Title |
exponential phase phase biological replicate 1 technical replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA bacterial growth in sixfors fermentor 1
|
| Organism |
Lactobacillus johnsonii |
| Characteristics |
Strain : NCC533
exponential phase 1
|
| Treatment protocol |
Aliquots of 15 ml for mid-exponential phases and 10 ml for adaptation and mid-stationary phases
Centrifuged for 5 min at 10,000 x g at 4°C.
Cell pellets were snap frozen in liquid nitrogen and stored at –80°C until further use.
|
| Growth protocol |
Grown in MRS with 2% of glucose as carbon source to which filter-sterilized cysteine was added after autoclaving (at a final concentration of 0.05% w/v) as redox buffer.
Cultures were incubated at 37°C under anaerobic conditions
cells were grown in a Sixfors fermentor system, composed of four individual 500 ml vessels (Infors, Bottmingen, Switzerland)
Four separate fermentation vessels (fermentor 1 to 4), which were inoculated at 0.4% (v/v) with four individual overnight cultures in order to reach a starting optical density (OD600nm) of 0.05.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were crushed and suspended in equal volumes of TE buffer (pH 8) and phenol (pH 4.2)
total RNA was extracted by the Macaloid method
The cells were disrupted at maximum speed using a Mini-Bead Beater-8 (BioSpec Products, Bartlesville, USA) at 4°C, during 1 min for three cycles with a resting period of 1 min on ice between each cycle
RNA was purified by phenol/chloroform extraction followed by ethanol precipitation
Pellets were re-suspended in nucleotide free water and 100 μg of total RNA was treated with 200 units of DNase I (Ambion, Huntingdon, United Kingdom) during 2 hours at 37°C to eliminate contaminating DNA.
The Rneasy mini-kit (Qiagen, Basel, Switzerland) that was used for further purification includes an additional on-column DNase digestion.
RNA concentrations were determined spectrophotometrically.
RNA integrity for all samples was tested using the Agilent RNA 6000 Nano assay (Agilent, Waldbronn, Germany).
|
| Label |
Cy5
|
| Label protocol |
2 μg of total RNA was labeled using the 3DNA Array 350RP Genisphere kit (Genisphere Inc., Hatfield, U.S.A)
Luciferase control mRNA (10 ng) (Promega, Zurich, Switzerland) was mixed with total RNA before labeling to balance the two channels during scanning
|
| |
|
| Channel 2 |
| Source name |
Total RNA bacterial growth reference (exponential phase)
|
| Organism |
Lactobacillus johnsonii |
| Characteristics |
Strain : NCC533
exponential phase 4
|
| Treatment protocol |
Aliquots of 15 ml for mid-exponential phases and 10 ml for adaptation and mid-stationary phases
Centrifuged for 5 min at 10,000 x g at 4°C.
Cell pellets were snap frozen in liquid nitrogen and stored at –80°C until further use.
|
| Growth protocol |
Grown in MRS with 2% of glucose as carbon source to which filter-sterilized cysteine was added after autoclaving (at a final concentration of 0.05% w/v) as redox buffer.
Cultures were incubated at 37°C under anaerobic conditions
cells were grown in a Sixfors fermentor system, composed of four individual 500 ml vessels (Infors, Bottmingen, Switzerland)
Four separate fermentation vessels (fermentor 1 to 4), which were inoculated at 0.4% (v/v) with four individual overnight cultures in order to reach a starting optical density (OD600nm) of 0.05.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were crushed and suspended in equal volumes of TE buffer (pH 8) and phenol (pH 4.2)
total RNA was extracted by the Macaloid method
The cells were disrupted at maximum speed using a Mini-Bead Beater-8 (BioSpec Products, Bartlesville, USA) at 4°C, during 1 min for three cycles with a resting period of 1 min on ice between each cycle
RNA was purified by phenol/chloroform extraction followed by ethanol precipitation
Pellets were re-suspended in nucleotide free water and 100 μg of total RNA was treated with 200 units of DNase I (Ambion, Huntingdon, United Kingdom) during 2 hours at 37°C to eliminate contaminating DNA.
The Rneasy mini-kit (Qiagen, Basel, Switzerland) that was used for further purification includes an additional on-column DNase digestion.
RNA concentrations were determined spectrophotometrically.
RNA integrity for all samples was tested using the Agilent RNA 6000 Nano assay (Agilent, Waldbronn, Germany).
|
| Label |
Cy3
|
| Label protocol |
2 μg of total RNA was labeled using the 3DNA Array 350RP Genisphere kit (Genisphere Inc., Hatfield, U.S.A)
Luciferase control mRNA (10 ng) (Promega, Zurich, Switzerland) was mixed with total RNA before labeling to balance the two channels during scanning
|
| |
|
| |
| Hybridization protocol |
Total RNA was hybridized onto DNA-arrays together with RNA extracted from mid-exponential phase cells
|
| Scan protocol |
array slides were scanned with a Scanarray 4000 machine (Packard Biochip Technologies, Billerica, U.S.A)
|
| Description |
exponential phase phase biological replicate 1 technical replicate 1
|
| Data processing |
Data were extracted from the scanned images using the software Imagene 5.6 (Biodiscovery, El Segundo, U.S.A).
Spots displaying low intensity (i.e. less than three fold the local background standard deviation) were considered empty (value=0)
For all technical replicates, the control for fluorescence labeling by dye-swap was done. As the method is based on qualitative detection, negative and positive controls were used to confirm the absence of cross and/or non-specific hybridization.
the absolute expression pattern was determined at least from three independent biological samples.
A gene was called as “expressed” (value=1) when it was detected in all three experiments.
|
| |
|
| Submission date |
Oct 04, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Emmanuel Denou |
| E-mail(s) |
emmanuel.denou@rdls.nestle.com
|
| Phone |
+ 41 21 785 9315
|
| Fax |
+ 41 21 785 8544
|
| Organization name |
Nestlé Research Center
|
| Department |
Nutrition & Health
|
| Lab |
Food & Health Microbiology
|
| Street address |
vers chez les blancs
|
| City |
Lausanne |
| State/province |
vaud |
| ZIP/Postal code |
PO Box 44-CH-1000 Lausanne 26 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL5958 |
| Series (1) |
| GSE9236 |
In vitro expression profiles of Lactobacillus johnsonii NCC533 in MRS |
|
