In order to study the function of the Campylobacter jejuni Cj1103 gene, a series of experiments were carried out. Three strains were constructed: a Cj1103 knockout strain, a strain where the Cj1103 knockout was complemented in trans, and a strain with a second copy over-expressing Cj1103 from an metK 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.
Overall design: 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).
Two or three independent RNA preparations (biological replicates) 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).
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