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Sample GSM560756 Query DataSets for GSM560756
Status Public on Jun 28, 2011
Title Ground Sample - biological replicate 3
Sample type RNA
 
Source name Ground sample - control
Organism Pseudomonas aeruginosa PAO1
Characteristics strain: PAO1
culture: bacteria cultured in normal ground conditions
atcc: ATCC 15692
Biomaterial provider American Type Culture Collection (ATCC 15692)
Treatment protocol A derivative of the wild type P. aeruginosa PAO1 (ATCC 15692) was used for the spaceflight experiment, which contained a gentamicin resistance cassette. The gentamicin-resistant strain was constructed through homologous recombination as described previously (Schweizer, 1993). P. aeruginosa PAO1 was grown in LB medium containing 25 µg/ml gentamicin in the spaceflight hardware (see below) to avoid growth of any contaminants. The bacterial inoculum 1.5 x 108 CFU/ml in the spaceflight hardware was suspended in 0.5 ml phosphate buffered saline (PBS) (Invitrogen), and remained viable but -static during launch and until nine days into the flight. Here after, growth was initiated by the addition of LB as described below. Cells were fixed in-flight using the RNA and protein fixative RNA Later II (Ambion). At 2.5 hours after Shuttle landing at the Kennedy Space Center (KSC), samples were recovered and subsequently used for whole genome transcriptional microarray and proteomic analysis. In each case, the flight culture samples were compared with culture samples grown under identical conditions on the ground at KSC using coordinated activation and termination times (by means of real-time communications with the Shuttle astronauts) in an insulated room that maintained identical temperature and humidity levels as those on the Shuttle (Orbital Environment Simulator).
Growth protocol P. aeruginosa PAO1 was grown in LB medium containing 25 µg/ml gentamicin in the spaceflight hardware (see below) to avoid growth of any contaminants. The bacterial inoculum 1.5 x 10+8 CFU/ml was suspended in 0.5 ml phosphate buffered saline (PBS)
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the RNeasy minikit (Qiagen) per the manufacturer’s instructions.
Label biotin
Label protocol Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression protocol. Briefly, random hexamers (Invitrogen) were added to 10 microgram of RNA along with
in vitro-transcribed Bacillus subtilis control spikes. cDNA was synthesized using Superscript III (Invitrogen) and the following conditions: 25°C for 10 min, 37°C for 60 min, and 70°C for 10 min. RNA was removed by alkaline treatment and subsequent neutralization. The cDNA was purified by using a QIAquick PCR purification kit (QIAGEN) and eluted in 40 microl of elution buffer (QIAGEN). The cDNA was then fragmented by using 0.6 U DNase I (Amersham) per microg cDNA
at 37°C for 10 min. The fragmented cDNA was end labeled with biotin-ddUTP by using a BioArray terminal labeling kit (Enzo) per the manufacturer’s instructions. A gel shift mobility assay was performed using NeutrAvadin (Pierce) on a 5% polyacrylamide gel stained with SYBR green (Roche) to ensure complete fragmentation and labeling.
 
Hybridization protocol Samples were hybridized, washed, stained, and scanned as described in the Affymetrix GeneChip P. aeruginosa genome array
expression analysis protocol.
Scan protocol Samples were hybridized, washed, stained, and scanned as described in the Affymetrix GeneChip P. aeruginosa genome array
expression analysis protocol.
Description gene expression data from bacteria cultured in ground conditions
Data processing Raw Affymetrix data were pre-processed using the Affy package in BioConductor as follows: (i) background correction based on the robust multichip average (RMA) convolution model (Irizarry et al., 2003), (ii) quantile normalization to make expression values from different arrays more comparable (Bolstad et al., 2003), (iii) summarization of multiple probe intensities for each probe set to one expression value per gene using RMA (Irizarry et al., 2003). To test for differential expression between the spaceflight and ground control cultures, the Bayesian adjusted t-statistics was used as implemented in the LIMMA package (version 2.5.15) (Smyth, 2004). P-values were corrected for multiple testing using the Benjamini and Hochberg’s method to control the false discovery rate (Benjamini and Hochberg, 1995). Microarray analysis was performed on all three biological replicates.
 
Submission date Jun 28, 2010
Last update date Jun 29, 2011
Contact name Rob Van Houdt
E-mail(s) rvhoudto@sckcen.be
Phone +3214332728
Organization name SCK-CEN
Department Interdisciplinary Biosciences
Lab Microbiology Unit
Street address Boeretang 200
City Mol
ZIP/Postal code 2400
Country Belgium
 
Platform ID GPL84
Series (1)
GSE22684 Transcriptional and proteomic response of Pseudomonas aeruginosa PAO1 to spaceflight conditions involves Hfq regulation and reveals a role for oxygen

Data table header descriptions
ID_REF
VALUE normalized log2 intensity signal

Data table
ID_REF VALUE
AFFX-Athal_actin_at 2.10328365476585
AFFX-Athal_GAPDH_at 2.10328365476585
AFFX-Athal_ubq_at 2.10328365476585
AFFX-Bsubtilis_dapB_at 7.37765366176357
AFFX-Bsubtilis_lys_at 2.10328365476585
AFFX-Bsubtilis_pheB_at 4.20623528266696
AFFX-Bsubtilis_thrC_at 5.53275629652632
AFFX-Bsubtilis_trpD_at 2.10328365476585
AFFX-YEL002C_WPB1_at 2.10328365476585
AFFX-YEL018W_at 2.10328365476585
AFFX-YEL024W_RIP1_at 2.10328365476585
AFFX-YER022W_SRB4_at 2.10328365476585
AFFX-YER148W_SPT15_at 2.10328365476585
AFFX-YFL039C_ACT1_at 2.10328365476585
ig_1046911_1047549_at 3.39852577775422
ig_1047549_1046911_at 6.54749601726527
ig_1063544_1064555_at 6.7352036511821
ig_1064555_1063544_at 3.3220533394223
ig_1087095_1087843_at 3.72084311638267
ig_1087843_1087095_at 5.63490190647253

Total number of rows: 5900

Table truncated, full table size 165 Kbytes.




Supplementary file Size Download File type/resource
GSM560756.CEL.gz 709.9 Kb (ftp)(http) CEL
Processed data included within Sample table

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