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Sample GSM482106 Query DataSets for GSM482106
Status Public on Jul 04, 2010
Title 240 h vs 82 h replicate a
Sample type RNA
 
Channel 1
Source name R. baltica cells 82 h
Organism Rhodopirellula baltica
Characteristics time point: Cells harvested after 82 h of growth
Treatment protocol Cultures were harvested by centrifugation (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor, 20 min, 6000 rpm, 4°C), after different incubation times (44, 62, 82, 96 and 240 h). The culture broth was collected in 500 ml tubes and swirled briefly in an ethanol/dry ice bath to rapidly cool down the cultures and freeze the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCl and then re-centrifuged to obtain cell pellets that were subsequently shock-frozen in liquid nitrogen and stored at -80°C
Growth protocol Cells of R. baltica were grown in batch culture (500ml) with mineral medium. The cultures were incubated at 28°C on a rotary shaker
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 Alexa546
Label protocol cDNA was directly labeled using the PlatinumBrightTM nucleic acid labeling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol
 
Channel 2
Source name R. baltica cells 240 h
Organism Rhodopirellula baltica
Characteristics time point: Cells harvested after 240 h of growth
Treatment protocol Cultures were harvested by centrifugation (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor, 20 min, 6000 rpm, 4°C), after different incubation times (44, 62, 82, 96 and 240 h). The culture broth was collected in 500 ml tubes and swirled briefly in an ethanol/dry ice bath to rapidly cool down the cultures and freeze the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCl and then re-centrifuged to obtain cell pellets that were subsequently shock-frozen in liquid nitrogen and stored at -80°C
Growth protocol Cells of R. baltica were grown in batch culture (500ml) with mineral medium. The cultures were incubated at 28°C on a rotary shaker
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 labeled using the PlatinumBrightTM nucleic acid labeling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol
 
 
Hybridization protocol Blocking, denaturing, hybridization, washing and drying of the slides with N2 were carried out in an automated hybridization station HS400 (Tecan, Crailsheim, Germany). The spotted arrays were blocked in prehybridization 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-labeled and 2 μg of Alexa 647 dye-labeled total cDNA were combined and taken up in a final volume of 100 µl DIG Easy Hyb hybridization solution (Roche Diagnostics, Mannheim, Germany). After the blocking step the sample solution was applied to the arrays, denaturized at 95°C for 3 min and hybridized under stringent conditions at 52°C for over 12 hours. After hybridization 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 Replicate_a
Data processing Raw data were automatically processed using the in-house 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 44 h / result of channel 62 h)) and I is the log mean fluorescence intensity (e.g. I = log10 (result of channel 44 h * result of channel 62 h)). 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. Normalization was carried out by LOWESS fitting on an R-versus-I plot with a smoothing factor of 0.05. Each time point of the growth curve experiment was hybridized. The expression data (ratio) of more than one hybridization were combined to one expression data point (ratio) by average and the standard deviation was calculated. 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 hybridization.
 
Submission date Dec 09, 2009
Last update date Feb 26, 2010
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)
GSE19405 Life cycle analysis of the model organism Rhodopirellula baltica by transcritpome studies

Data table header descriptions
ID_REF
VALUE lowess normalized ratio ch1/ch2 (control/experimental)

Data table
ID_REF VALUE
pc_RB10279_A -1.276088791
pc_RB10279_A_70
pc_RB10279_A_80
pc_RB10279_A_90
pc_RB10279_B -1.901027327
pc_RB10279_B_70 -1.545715254
pc_RB10279_B_80
pc_RB10279_B_90
pc_RB10279_C -1.662904725
pc_RB10279_C_70 -0.128811177
pc_RB10279_C_80 -0.542500028
pc_RB10279_C_90 0.161141129
pc_RB10279_D_70 -0.467529303
pc_RB10279_D_80 -0.475397105
pc_RB10279_D_90 -1.980878368
pc_RB10279_E_70 0.673096219
pc_RB10279_E_80
pc_RB10279_E_90 1.983019022
pc_RB10279_F_70 -1.93745304
pc_RB10279_F_80 -0.493921158

Total number of rows: 7733

Table truncated, full table size 141 Kbytes.




Supplementary file Size Download File type/resource
GSM482106.txt.gz 2.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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