|
| Status |
Public on Feb 13, 2008 |
| Title |
L.reuteri_sourdough_rep1_dyeswap2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L.reuteri, exponential phase, sourdough
|
| Organism |
Limosilactobacillus reuteri |
| Characteristics |
strain ATCC 55730
|
| Treatment protocol |
L. reuteri was grown in sourdough and CDML until exponential growth phase.
|
| Growth protocol |
Chemically defined medium CDML and sourdough (both containing 10µg/g erythromycin) were inoculated with over night cultures of L. reuteri: 1% inoculation of ripe dough and 1:100 inoculation of CDML culture, respectively. Both media were then incubated at 40°C for 3.5 h (sourdough) and to an OD600 value of 0.3 (CDML).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from L. reuteri was isolated using the Gentra Purescript RNA Isolation Kit (Gentra Systems) with modifications. L. reuteri cells (approximately 1 x 10^9 cells) were resuspended in 200 µL TE buffer containing 20 mg/ml lysozyme (SERVA) and 500 U/ml mutanolysin (Sigma-Aldrich) and incubated for 30 minutes at 37 °C. The cell suspensions were transferred to 2.0 ml screw cap tubes containing 50 mg of 0.1-mm zirconia beads (Carl Roth), and 200 µL Cell Lysis Solution (Gentra) was added. The cells were disrupted at 5,000 rpm for 5 min using the Mini-Beadbeater (BioSpec Products). 100 µL DNA/Protein Precipitation Solution (Gentra Systems) was added to the lysate and the mixture was incubated at 65 °C for 5 min. After centrifugation, the clear supernatant containing the RNA was added to 300 µL isopropanol and total RNA was harvested by centrifugation. After two washes with 70% ethanol the RNA was resuspended in RNAse-free deionized water and storage at –85 °C. The samples were treated with DNAse I (Roche Applied Science) and subsequently purified with RNeasy Mini Kit (Qiagen), both according to the manufacturers' instructions. The concentration and purity of RNA samples were determined by standard spectrophotometer measurements and agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
10 ug of total RNA were primed with 2.5 ug of random hexamers (Qiagen) at 70 degrees Celsius for 10 minutes. Reverse transcription was then carried out using Superscript III (Invitrogen) with 500 uM each dATP, dCTP, and dGTP, 300 uM dTTP, and 200 uM aminoallyl dUTP. After reverse transcription, RNA was removed by hydrolysis and cDNA was cleaned. cDNA was then labeled using CyDye Post-labeling reactive dye packs (Amersham).
|
| |
|
| Channel 2 |
| Source name |
L.reuteri, exponential phase, CDML
|
| Organism |
Limosilactobacillus reuteri |
| Characteristics |
strain ATCC 55730
|
| Treatment protocol |
L. reuteri was grown in sourdough and CDML until exponential growth phase.
|
| Growth protocol |
Chemically defined medium CDML and sourdough (both containing 10µg/g erythromycin) were inoculated with over night cultures of L. reuteri: 1% inoculation of ripe dough and 1:100 inoculation of CDML culture, respectively. Both media were then incubated at 40°C for 3.5 h (sourdough) and to an OD600 value of 0.3 (CDML).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from L. reuteri was isolated using the Gentra Purescript RNA Isolation Kit (Gentra Systems) with modifications. L. reuteri cells (approximately 1 x 10^9 cells) were resuspended in 200 µL TE buffer containing 20 mg/ml lysozyme (SERVA) and 500 U/ml mutanolysin (Sigma-Aldrich) and incubated for 30 minutes at 37 °C. The cell suspensions were transferred to 2.0 ml screw cap tubes containing 50 mg of 0.1-mm zirconia beads (Carl Roth), and 200 µL Cell Lysis Solution (Gentra) was added. The cells were disrupted at 5,000 rpm for 5 min using the Mini-Beadbeater (BioSpec Products). 100 µL DNA/Protein Precipitation Solution (Gentra Systems) was added to the lysate and the mixture was incubated at 65 °C for 5 min. After centrifugation, the clear supernatant containing the RNA was added to 300 µL isopropanol and total RNA was harvested by centrifugation. After two washes with 70% ethanol the RNA was resuspended in RNAse-free deionized water and storage at –85 °C. The samples were treated with DNAse I (Roche Applied Science) and subsequently purified with RNeasy Mini Kit (Qiagen), both according to the manufacturers' instructions. The concentration and purity of RNA samples were determined by standard spectrophotometer measurements and agarose gel electrophoresis.
|
| Label |
Cy5
|
| Label protocol |
10 ug of total RNA were primed with 2.5 ug of random hexamers (Qiagen) at 70 degrees Celsius for 10 minutes. Reverse transcription was then carried out using Superscript III (Invitrogen) with 500 uM each dATP, dCTP, and dGTP, 300 uM dTTP, and 200 uM aminoallyl dUTP. After reverse transcription, RNA was removed by hydrolysis and cDNA was cleaned. cDNA was then labeled using CyDye Post-labeling reactive dye packs (Amersham).
|
| |
|
| |
| Hybridization protocol |
For hybridization, yeast tRNA and salmon testes DNA were added to labeled cDNA along with a formamide hybridization buffer. Samples were applied to slides, and hybridization was carried out at 42 degrees Celsius in Corning microarray hybridization chambers overnight. Slides were then washed sequentially for four minutes in each of the following buffers: 1X SSC and 0.2% SDS, 0.1X SSC and 0.2% SDS, and 0.1X SSC.
|
| Scan protocol |
Arrays were scanned on a Axon instruments GenePix 4000B scanner. Images were processed using GenePix Pro software (version 4.1)
|
| Description |
A comparison of Lactobacillus reuteri cells grown in chemically defined medium (CDML) and sourdough helped uncover pathways involved in the adaptation of L. reuteri to the ecological niche of sourdough.
|
| Data processing |
Only spots with >= 40% of pixels with fluorescence value > 1 standard deviation above the local background in at least one of the channels were considered valid. Normalization: Total fluorescence normalization. Calculation of expression ratios: fluorescence values of sourdough samples/values of CDML samples. Only genes that displayed valid values in at least two of the three biological replicates were used for further analysis. The geometric mean of the expression ratios and standard deviations were calculated and log2-transformed. For each gene, the ratio was generated using the average of three independent experiments (independently grown and prepared samples) with two replicate hybridizations. Genes with M-values (mean log2 ratio RNAdough / RNACDML) smaller than –1 (<0.5-fold expression) were considered repressed and values greater that +1 (>2-fold expression) were considered induced in sourdough as compared to CDML medium. To determine which genes differed statistically from the mean, we utilized iterative outlier analysis (Britton et al., 2002), in which three successive rounds of analysis are done to find genes with M-values greater than 2.5 standard deviations from the mean. Confidence limits of the data were calculated, including a standard error factor (distance from gene's average value to the nearest 2.5 standard deviation cut-off) and the number of standard deviations separating the spot from the 2.5 standard deviation cut-off. The M-values of outliers ranged from 5.08 (33.7-fold change) to -3.75 (0.07-fold change). Furthermore, all genes belonging to a putative operon were considered for analysis if at least one gene of the operon showed significant changes in the expression, and the remaining genes showed trends toward that expression.
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| |
|
| Submission date |
Feb 12, 2008 |
| Last update date |
Feb 12, 2008 |
| Contact name |
Eric Huefner |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Hohenheim
|
| Department |
Food Microbiology
|
| Street address |
Garbenstrasse 28
|
| City |
Stuttgart |
| ZIP/Postal code |
70599 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6366 |
| Series (1) |
| GSE10495 |
Global transcriptome analysis of Lactobacillus reuteri in sourdough fermentation. |
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