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| Status |
Public on Sep 09, 2009 |
| Title |
R. baltica_salt shift_10min_a |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rhodopirellula baltica salt shift reference
|
| Organism |
Rhodopirellula baltica |
| Characteristics |
non-treated = reference = time point zero
|
| Treatment protocol |
In all cases, cells were grown to the OD600nm = 0.5 (corresponding log phase), and an aliquot of cells was collected to serve as the time-zero reference. The culture broth was collected in 500 mL tubes and swirled briefly in an ethanol-dry ice bath to rapidly cool the cultures and "freeze" the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman CoulterTM AvantiTM626 J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged to cell pellets that were shock-frozen in liquid nitrogen and stored at -80°C
|
| Growth protocol |
For all experiments Rhodopirellula baltica SH 1T cells were grown as chemostate cultures in a mineral medium containing 10 mM glucose as sole carbon and 1 mM ammonium chloride as nitrogen source at 28°C (Rabus et al. 2002)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The culture pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged to cell pellets that were shock-frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using the proposed protocol of the TRI Reagent® Kit by Ambion (Austin, USA).
cDNA synthesis was performed using the SuperScript direct cDNA labeling system kit by Invitrogen (Karlsruhe, DE) according to the manufacturer's instructions with random hexamers and unlabeled dCTP/dUTP.
|
| Label |
Alexa 546
|
| Label protocol |
cDNA was directly labelled using the PlatinumBrightTM nucleic acid labelling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol.
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| |
|
| Channel 2 |
| Source name |
Rhodopirellula baltica, salt shift, 10min
|
| Organism |
Rhodopirellula baltica |
| Characteristics |
Salt shifted for 10min
Replicate a
|
| Treatment protocol |
Cells grown continuously at 28°C and in a medium with 17.5% salinity were collected by centrifugation. Aliquots were resuspend in an equal volume of medium containing 59.5% salinity and returned for growth. Samples were collected at 10, 20, 40, 60 and 300 min
|
| Growth protocol |
For all experiments Rhodopirellula baltica SH 1T cells were grown as chemostate cultures in a mineral medium containing 10 mM glucose as sole carbon and 1 mM ammonium chloride as nitrogen source at 28°C (Rabus et al. 2002)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The culture pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged to cell pellets that were shock-frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using the proposed protocol of the TRI Reagent® Kit by Ambion (Austin, USA).
cDNA synthesis was performed using the SuperScript direct cDNA labeling system kit by Invitrogen (Karlsruhe, DE) according to the manufacturer's instructions with random hexamers and unlabeled dCTP/dUTP.
|
| Label |
Alexa647
|
| Label protocol |
cDNA was directly labelled using the PlatinumBrightTM nucleic acid labelling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Blocking, denaturing, hybridisation, washing and drying of the slides with N2 were carried out in an automated hybridisation station HS400 (Tecan, Crailsheim, Germany). The spotted arrays were blocked in prehybridisation solution containing 250 mM NaCl, 5 mM Tris/HCl at pH 8.0, 50% formamide, 0.5x SSC, 0.05% BSA, and 1% blocking reagent from Roche Diagnostics, Mannheim, Germany for 45 min at 52°C. For hybridisation at least 2 μg of Alexa 546 dye-labelled and 2 μg of Alexa 647 dye-labelled total cDNA were combined and taken up in a final volume of 100 µl DIG Easy Hyb hybridisation solution (Roche Diagnostics, Mannheim, Germany). After the blocking step the sample solution was applied to the arrays, denaturised at 95°C for 3 min and hybridised under stringent conditions at 52°C for over 12 hours. After hybridisation slides were washed at RT in ULTRArray Low Stringency Wash Buffer (Ambion, Austin, USA) and dried by N2.
|
| Scan protocol |
Slides were scanned at a resolution of 5 μm using a ScanArray Express Microarray scanner (Perkin Elmer, Wellesley, USA) with varied laser power and sensitivity level of the photomultiplier tube (PMT) for each slide. The provided image analysis software ScanArray Express Version 4.0 was used for automatic spot detection and signal quantification of both fluorophores.
|
| Description |
salt shift 10min vs. reference
|
| Data processing |
Raw data were automatically processed using the inhouse developed microarray data analysis software tool MADA (www.megx.net/mada). First of all the spot intensities were local background corrected (spot intensity minus spot background intensity). Then signals were only assessed as positive if mean spot pixel intensity was higher than the mean local background intensity plus 2 times the standard deviation of the mean local background pixel intensity. Each gene is spotted in 3 replicates. Spot replicates with poor quality were removed from the data set according to the outlier test of MADA. For this test, first the standard deviation of all replicates is computed. Second, one replicate is omitted and the standard deviation is recalculated, if the deviation differs more than 50% from the previous deviation, the omitted replicate is regarded as outlier. This procedure is alternately repeated for all replicates
The expression is described through the ratio and intensity, where R is the fluorescence log ratio of the experiment time point relative to the control condition (e.g. R = log2 (result of channel 10min / result of channel control/reference)) and I is the log mean fluorescence intensity (e.g. I = log10 (result of channel 10min * result of channel control/reference)).
Each data point represents a regulation factor (ratio) in a logarithmic scale for one gene calculated from the positive replicates for a particular probe coming from two RNA pools (reference and sample). Normalisation was carried out by LOWESS fitting on an R-versus-I plot with a smoothing factor of 0.05. Each time point of the time-series experiment was hybridised independently three times. The expression data (ratio) of the three hybridisations were combined to one expression data point (ratio) by average and the standard deviation was calculated. Only ratios with a standard deviation less than 25% were taken as regulated.
Differentially expressed genes are determinate by a fixed threshold cut off method (i.e. a two-fold increase or decrease) based on the self-self hybridisation.
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| |
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| Submission date |
Dec 20, 2008 |
| Last update date |
Sep 09, 2009 |
| Contact name |
Patricia Wecker |
| E-mail(s) |
pwecker@mpi-bremen.de
|
| Phone |
00494212028982
|
| Organization name |
Max Planck Institut for Marine Microbiology
|
| Department |
Molecular Ecology
|
| Lab |
Microbial Genomics Group
|
| Street address |
Celsiusstr, 1
|
| City |
Bremen |
| ZIP/Postal code |
28359 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7654 |
| Series (1) |
| GSE14075 |
Response of Rhodopirellula baltica to salt shift |
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