|
| Status |
Public on May 25, 2012 |
| Title |
pNZ8901 vs pNZ-nprE_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Net intensity (tr.mean) {A}
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype/variation: Wild type
strain: NZ8900
plasmid: pNZ8901
group: control
|
| Treatment protocol |
When cells reached OD600=0.7, 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). 30 minuts after subtilin induction, 10 OD units of each culture were collected for total RNA isolation (1 OD unit = 1 ml of a culture of an OD600 = 1.0).
|
| Growth protocol |
50 ml of LB was inoculated with over/night cultures to an OD600=0.05 and cultures were grown under vigurous shaking (225 rpm) at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) according to the manufacturer's instruction. Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three or four replicate cultures.
|
| Label |
Cy3
|
| Label protocol |
20 μg of 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 CyScribe GFX purification kit (Amersham Biosciences).
|
| |
|
| Channel 2 |
| Source name |
Net intensity (tr.mean) {B}
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype/variation: Overexpression mutant
strain: NZ8900
plasmid: pNZ-nprE
group: test
|
| Treatment protocol |
When cells reached OD600=0.7, 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). 30 minuts after subtilin induction, 10 OD units of each culture were collected for total RNA isolation (1 OD unit = 1 ml of a culture of an OD600 = 1.0).
|
| Growth protocol |
50 ml of LB was inoculated with over/night cultures to an OD600=0.05 and cultures were grown under vigurous shaking (225 rpm) at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) according to the manufacturer's instruction. Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three or four replicate cultures.
|
| Label |
Cy5
|
| Label protocol |
20 μg of 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 CyScribe GFX purification kit (Amersham Biosciences).
|
| |
|
| |
| Hybridization protocol |
The labelled cDNA was hybridized to oligo DNA microarray in Ambion Slidehyb #1 buffer (Ambion Eurpe Ltd) at 48°C for 18 hours. Next, microarray slides were washed in 2 × SSC with 0.5% SDS, 1 × SSC with 0.25% SDS and 1 × SSC with 0.1% SDS and dried by centrifugation.
|
| Scan protocol |
Images were acquired with GeneTac LS IV confocal laser scanner (Genomics Solutions).
|
| 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 (van Hijum et. al. (2003) Appl. Bioinformatics 2, 241-244). 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 overexpression 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 |
Dec 16, 2011 |
| Last update date |
May 25, 2012 |
| Contact name |
Bogumila Marciniak |
| Organization name |
Rijksuniversiteit Groningen
|
| Department |
Department of Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15025 |
| Series (1) |
| GSE34505 |
Comparative transcriptional analysis of bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins |
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