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| Status |
Public on May 23, 2007 |
| Title |
Lactobacillus brevis and Pectinatus frisingensis detected by microarray |
| Sample type |
RNA |
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| Source name |
Beer spoilage bacteria
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| Organisms |
Pectinatus frisingensis; Levilactobacillus brevis |
| Characteristics |
Lactobacillus brevis Isolate from Beck & Co., Bremen, Germany, Pectinatus frisingensis DSM 6306 (T)
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| Biomaterial provider |
Beck & Co., Bremen, Germany, DSMZ
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| Growth protocol |
Lactobacillus brevis was cultured at 30°C in De Man-Rogosa-Sharpe MRS medium. Pectinatus frisingensis was cultured under anaerobic conditions at 30°C in peptone-yeast extract plus Fildes solution medium.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Portions (40 ml) of exponentially growing cultures (OD600 of ~0.8) were chilled on ice and cells were harvested immediately by centrifugation (10 min, 300 x g, 4°C). All plastic wares were treated with 0.1% DEPC at 37°C over night and autoclaved. Total RNA was isolated according to Hurt et al. (2001) with several modifications. All steps were performed at 4°C. The samples were saturated with 170 μl denaturation solution containing 0.85 μl 2-mercaptoethanol and 1.5 ml extraction buffer and incubated for 30 min at 65°C, gently mixing and centrifuged at 1800 x g for 10 min. The supernatants were poured into tubes and chilled on ice. The pellet was resuspended in 830 μl extraction buffer, incubated for 10 min at 65°C gently mixing and centrifugated at 1800 x g for 10 min. The supernatants were combined, the same volume of phenol-chloroform was added and the sample was centrifugated at 1800 x g for 20 min. The aqueous phase was transferred to a new tube and precipated with 0.6 volumes ice cold ethanol (100%) at -20°C over night. The sample was centrifugated at 16000 x g for 20 min, washed with ice cold ethanol (70%) and centrifugated at 16000 x g for 10 min. The pellet was dried at 4°C and resuspended in 200 μl H2O. RNA concentration was determined by measurement the optical density at 260 nm (OD260).
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| Label |
Cy3
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| Label protocol |
The cDNA was labeled with Cy3-fluorescent dyes using the Mirus Label IT-Kit (MoBiTec, Göttingen, Germany) according to the manufacturer’s instructions.
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| Hybridization protocol |
1 μg Cy3-labeled cDNA was mixed with 3 μl 20x SSC, 0.5 μl 1 M HEPES (pH 7) and 0.5 μl 10% SDS, heated to 99°C for 2 min, chilled on ice, incubated at room temperature for 5 min, applied to the microarrays which were covered with a lifterslip (Erie Scientific Company, Portsmouth, USA) and incubated for 12 to 16 hours at 30°C in a moist chamber (1x SSC). After hybridization the microarrays were washed in a solution containing 2x SSC and 0.1% SDS, then in 1x SSC and then in 0.5x SSC. After washing microarrays were dried by centrifugation (15 min at 50 x g).
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| Scan protocol |
After drying the microarrays the fluorescence at 532 nm (Cy3) was determined using an Axon Inc. GenePix 4000 laser scanner (Axon, Union City, USA). Acquired raw fluorescence data were stored as image files in TIFF format and were analysed quantitatively by using GenePix Pro 3.0 software (Axon, Union City, USA).
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| Description |
RT-PCR amplification. All plastic wares were treated with 0.1% DEPC at 37°C over night and autoclaved. The RT-PCR amplification was carried out using T3 Thermocycler (Whatman Biometra, Göttingen, Germany). The RT reaction mixture contained 2.5 μl pd(N)6 random hexamer 5´-phosphate [500 ng/μl] (Roche, Darmstadt, Germany), 20 mM deoxynucleoside triphosphates, 0.5 μl T4 gene 32 protein [5 mg/ml] (Roche Diagnostics, Mannheim, Germany), 2 μl H2O and a maximum of 1.5 μg RNA. Samples were incubated at 65°C for 5 min, immediately chilled on ice and centrifuged. 4 μl First-strand buffer (Invitrogen, Carlsberg, USA), 2 μl 0.1 M DTT, 1 μl 2.5 mM MgCl2 and 40 U RNase-Inhibitor (Roche Diagnostics, Mannheim, Germany) were added to the mixture and incubated at 42°C for 2 min. Then, 200 U SuperScript II (Invitrogen, Carlsberg, USA) were added to the mixture and incubated at 42°C for 50 min followed by an incubation at 70°C for 15 min. An aliquot of 5 μl of the RT reaction mixture was used for PCR amplification. The PCR reaction mixture contained 5 μl 10x PCR buffer (Invitrogen, Carlsberg, USA), 2 mM deoxynucleoside triphosphates, 10 pmol of each primer (MWG AG Biotech, Ebersberg, Germany), 1.5 mM MgCl2, 5 U Taq-Polymerase (Invitrogen, Carlsberg, USA) in a total reaction volume of 50 μl. Samples were initially denatured at 94°C for 3 min, followed by 40 sequential cycles, denaturation at 95°C (1 min), annealing at 40°C (1 min), extension at 72°C (1 min) and a final extension at 72°C for 5 min followed by a cooling step down to 4°C. PCR products were analysed for correct size by agarose gel electrophoresis and ethidium bromide staining.
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| Data processing |
Signal was defined of median spot fluorescence signal at 532 nm wavelength (F532) minus median background fluorescence signal (B532), defined by surrounding pixel intensity and the mean value of 70 replicate spots was calculated.
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| Submission date |
May 18, 2007 |
| Last update date |
May 22, 2007 |
| Contact name |
Daniel Gilbert Weber |
| Organization name |
IPA
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| Department |
Molekulare Medizin
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| Street address |
Bürkle-de-la-Camp Platz 1
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| City |
Bochum |
| ZIP/Postal code |
44789 |
| Country |
Germany |
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| Platform ID |
GPL5158 |
| Series (1) |
| GSE7840 |
Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria |
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| Data table header descriptions |
| ID_REF |
|
| VALUE |
Mean value |
| Data table |
| ID_REF |
VALUE |
| 1 |
18220 |
| 2 |
228 |
| 3 |
454 |
| 6 |
1497 |
| 7 |
6494 |
| 8 |
186 |
| 9 |
22497 |
Total number of rows: 7
| Supplementary file |
Size |
Download |
File type/resource |
| GSM190248.gpr.gz |
29.0 Kb |
(ftp)(http) |
GPR |
| Processed data included within Sample table |
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