Mountain View
biomedical and healthCAre Data Discovery Index Ecosystem
help Advanced Search
Title: Study of Cj0138 function in Campylobacter jejuni      
dateReleased:
12-31-2012
description:
In order to study the function of the Campylobacter jejuni Cj0138 gene, a series of experiments were carried out. Three strains were constructed: a Cj0138 knockout strain, a strain where the Cj0138 knockout was complemented in trans, and a strain with a second copy over-expressing Cj0138 from an fdxA promoter. The transcriptomes of these were all compared to the wild-type strain. The arrays are all from RNA isolated in mid-exponential growth from independent biological replicates. Batch cultures of Campylobacter jejuni NCTC 11168 were grown in 50 ml volumes of Brucella broth in 70cm tissue culture flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (5% Oxygen, 10% Carbon dioxide, 85% Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 37 ºC. RNA was extracted from C. jejuni cultures grown to an OD600 of approximately 0.4. Briefly, 0.1 volume of 5% phenol in ethanol was mixed with the broth culture, and after centrifugation RNA was isolated with Tri Reagent (Sigma) and chloroform. RNA was further purified using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. The RNA was treated with Turbo DNA-free (Ambion) to remove any residual DNA according to the manufacturer’s instructions. RNA concentration was determined using the Nanodrop Spectrophotometer NS-1000 (Thermo Scientific). RNA preparations of each sample type were labelled and hybridized to custom-designed Agilent microarrays. Equal quantities of RNA from test and control cultures were labelled by using nucleotide homologues of dUTP containing either Cy3 or Cy5 fluorescent dye (Perkin Elmer). For each microarray slide, the test strain was labelled with Cy3-dUTP, while the wild-type sample was labelled with Cy5-dUTP. RNA (15 µg) was primed with 5 µg pd(N)6 random hexamers (Amersham Biosciences). The Affinity Script kit (GE Healthcare) was used to produce cDNA, and hybridized (Agilent Hi-RPM Gene Expression Hybridization Kit) to the microarray slide according to the manufacturer’s instructions. Microarrays were scanned at 5 µm using an Axon 4000A scanner, and images were acquired using GenePix Pro 3.0 software (Axon). Related analysis reference: Holmes K., Mulholland F., Pearson B. M., Pin C., McNicholl-Kennedy J., Ketley J. M., Wells J. M. (2005) Campylobacter jejuni gene expression in response to iron limitation and the role of Fur Microbiology-SGM 151 243-257 (PMID 15632442).
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-35119
refinement:
raw
alternateIdentifiers:
35119
keywords:
functional genomics
dateModified:
06-02-2014
availability:
available
types:
gene expression
name:
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819
ID:
A-GEOD-13841
name:
Agilent-019568 Campylobacter 11168 expression array_1_Of_1
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-35119/E-GEOD-35119.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-35119/E-GEOD-35119.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35119
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress