|
| Status |
Public on Feb 01, 2011 |
| Title |
Slide 188694 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Macrocolonies from rok
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype: rok
|
| Growth protocol |
Macrocolonies were grown on 2xSG plate and harveseted for RNA preparation after 3 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences
|
| |
|
| Channel 2 |
| Source name |
Macrocolonies from wt
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype: wt
|
| Growth protocol |
Macrocolonies were grown on 2xSG plate and harveseted for RNA preparation after 3 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences
|
| |
|
| |
| Hybridization protocol |
The protocol was performed as described in Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner
|
| Description |
rok vs wt replicate 4
|
| Data processing |
Dual-channel array images were analyzed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP as previously described (den Hengst et al J. Biol. Chem. 280:34332-34342; PMID: 16040604). Prior to analysis, automatically and manually flagged spots and spots with very low background subtracted signal intensity (5% of the weakest spots [sum of Cy3 and Cy5 net signals]) were filtered out of all data sets.Net signal intensities were calculated by grid-based background subtraction. A grid-based Lowess transformation was performed for slide normalization, negative and empty values were removed, and outliers were removed by the deviation test. Expression ratios of mutant strain over the wild-type strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.
|
| |
|
| Submission date |
Jun 15, 2010 |
| Last update date |
Feb 01, 2011 |
| Contact name |
Akos T Kovacs |
| Organization name |
Friedrich Schiller University of Jena
|
| Department |
Institute of Microbiology
|
| Lab |
Terrestrial biofilms
|
| Street address |
Neugasse 23
|
| City |
Jena |
| ZIP/Postal code |
07743 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6031 |
| Series (1) |
| GSE22370 |
Role of Rok in biofilm structure development of Bacillus subtilis 168 |
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