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Sample GSM435808 Query DataSets for GSM435808
Status Public on Aug 08, 2009
Title Logarithmic phase sample 4
Sample type RNA
 
Channel 1
Source name rpoN mutant grown to logarithmic phase
Organism Escherichia coli O157:H7 str. Sakai
Characteristics genotype: rpoN mutant (EcJR-8)
growth protocol: logarithmic phase in DMEM-MOPS
Growth protocol EcJR-8 grown to OD600 = 0.5 in DMEM-MOPS 0.4% glucose at a 10:1 flask-to-media volume in a rotary shaker (180 RPM).
Extracted molecule total RNA
Extraction protocol 5mL of culture was mixed with 5mL of hot acid phenol:chloroform. Samples were held at 65ºC with periodic shaking for at least 10 minutes before centrifuging at 4000 rpm for 20 min. Supernatant was extracted again with acid-phenol:chloroform and then with chloroform:isoamyl alcohol (24.1). RNA was precipitated overnight at –80ºC in 2.5V 100% ethanol and 1/10V 3M sodium acetate pH 5.2. RNA samples were purified and treated with DNase using the Rneasy kit (Qiagen).
Label Cy3
Label protocol Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM amino-allyl dUTP. 30uL C. cDNA was purified using PCRreactions were incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
 
Channel 2
Source name wild type grown to logarithmic phase
Organism Escherichia coli O157:H7 str. Sakai
Characteristics genotype: wild type
growth protocol: logarithmic phase in DMEM-MOPS
Extracted molecule total RNA
Extraction protocol 5mL of culture was mixed with 5mL of hot acid phenol:chloroform. Samples were held at 65ºC with periodic shaking for at least 10 minutes before centrifuging at 4000 rpm for 20 min. Supernatant was extracted again with acid-phenol:chloroform and then with chloroform:isoamyl alcohol (24.1). RNA was precipitated overnight at –80ºC in 2.5V 100% ethanol and 1/10V 3M sodium acetate pH 5.2. RNA samples were purified and treated with DNase using the Rneasy kit (Qiagen).
Label Cy5
Label protocol Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM amino-allyl dUTP. 30uL C. cDNA was purified using PCRreactions were incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
 
 
Hybridization protocol Arrays were cross-linked by exposure to 600 mJ UV before blocking in 1% SDS, 5× SSC, and 1 mg/mL BSA at 42°C for 1 hour. After blocking, arrays were washed 2× 5 min in 0.1× SSC and 2× 30 s in H2O. Dried arrays were placed into hybridization cassettes (TeleChem International, Sunnyvale, Calif.) and the cDNA samples were suspended in 10 mM EDTA, denatured at 95°C for 5 min and then mixed with 40 uL of SlideHyb buffer 1 (Ambion) and loaded under a coverslip onto the array. Hybridizations were carried out at 47°C for 16–18 h. After hybridization, arrays were washed in 2× SSC, 0.5% SDS 37C for 5 min, followed by 2× 5 min in 0.1× SSC, 0.1% SDS 37°C, and then 2 × 2.5 min in room temperature 0.1× SSC. Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Scan protocol Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Description There are probes on the array that target three different E. coli genomes. Those that were specific for genomes other than Sakai were excluded from the analysis.
Data processing The microarray data were analyzed using R (v. 2.2.1) and the MAANOVA (v. 0.98.8) package. Raw intensity values from replicate probes were averaged and log2 transformed after normalization with the pin-tip LOWESS method. The normalized intensity values were fitted to a mixed model ANOVA considering array and biological replicates as random factors and dye, strain and growth phase as fixed factors. The linear model tested was Y (intensity) = array + dye + strain (wild type or mutant) + growth phase (exponential or stationary) + strain*growth phase + sample (biological replicate) + error. Significant differences in expression due to strain, growth phase and strain*growth phase were determined using the Fs test in MAANOVA which uses a shrinkage estimator for gene-specific variance components that makes no assumption about the variances across genes with 500 random permutations to estimate the p-values. The q-value package in R was used for determining the false discovery rate (FDR).
 
Submission date Jul 31, 2009
Last update date Aug 07, 2009
Contact name James Timothy Riordan
E-mail(s) [email protected]
Organization name University of South Florida
Department CMMB
Lab Riordan Lab
Street address 4202 E Fowler Ave BSF 218
City Tampa
State/province FL
ZIP/Postal code 33620
Country USA
 
Platform ID GPL7445
Series (1)
GSE17467 The RpoN Regulon of Escherichia coli O157:H7

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (rpoN mutant/wt)
EcJR-8 normalized signal for rpoN mutant
Sakai normalized signal for WT

Data table
ID_REF VALUE EcJR-8 Sakai
E100000001 1.718884541 13.01891415 11.30002961
E100000002 -0.60376589 11.50413488 12.10790077
E100000003 -1.167620599 12.36391599 13.53153659
E100000004 -0.751233356 11.82879463 12.58002799
E100000005 -0.431026255 7.577974736 8.009000991
E100000006 -0.049840318 9.469302115 9.519142433
E100000007 0.073854505 8.312057424 8.238202919
E100000008 -0.342764407 12.79208083 13.13484523
E100000009 -0.251909255 11.7781686 12.03007786
E100000010 -0.199011597 9.15218917 9.351200767
E100000011 -0.107094637 8.958922915 9.066017552
E100000012 -0.192380351 9.195313913 9.387694264
E100000013 2.003236104 12.31601818 10.31278207
E100000014 -1.333423368 11.67783306 13.01125643
E100000015 -0.767209901 9.625963273 10.39317317
E100000016 -0.466433544 9.739953043 10.20638659
E100000017 -0.145278737 7.746682278 7.891961015
E100000018 0.707585272 16.11998922 15.41240395
E100000019 0.304792881 9.560064435 9.255271553
E100000020 0.557448213 11.0545854 10.49713718

Total number of rows: 6088

Table truncated, full table size 280 Kbytes.




Supplementary file Size Download File type/resource
GSM435808.gpr.gz 1.3 Mb (ftp)(http) GPR
Processed data included within Sample table

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