BioProject

Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. More...

Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.010 mM NOC-5 & NOC-7 was added to one of the cultures. After a 15 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Overall design: Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 µl phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 μg) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 μg pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 μl elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For control slide Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For dye-swap slide Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made for compensate possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChip® arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVac® (Thermo Savant, Waltham, MA, USA) The dry cDNA was resuspended in pre-warmed (42ºC) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 °C for 3 min and then placed on ice for 3 min. The spoted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42º C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37º C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc (Imagene, version 4.0 and Genesight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the Student’s t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated ≥ 2-fold and displaying a p-value of ≤ 0.05 were defined as being statistically significant and differentially transcribed. Less...