Campylobacter jejuni exposure to 0.25mM GSNO

Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 150 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 5/10/15/45 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. Keywords: Time Course Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 150 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. Two independent RNA preparations (biological replicates) of each sample were labelled and hybridized to glass-slide microarrays. Equal quantities of RNA from control and GSNO treated cultures were labelled by using nucleotide homologues of dCTP containing either Cy3 or Cy5 florescent dye (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while another sample was labelled with Cy5-dCTP. RNA (15 µg) was primed with 5 µg pd(N)6 random hexamers (Amersham Biosciences) and made to 12.4 µl total volume with water and supplemented on ice with 3 µl 10x RT buffer (Invitrogen), 0.6 µl 50x dNTPs mixture (25 mM each dATP, dTTP, dGTP and 10 mM dCTP), 3 µl 0.1 M DTT, 2 µl 1mM Cy3 or Cy5 (Perkin Elmer), and 200U Superscript II RNase H reverse transcriptase (Invitrogen). Incubation was at 37 °C overnight. Labelled cDNA was purified using a Qiaquick purification kit (Qiagen) and dried before being resuspended in 19.5 µl water, 2.25 µl human Cot1 DNA (Invitrogen), 4.5 µl 20x SSC, 0.72 µl 1 M HEPES, pH 7.0, 0.68 µl 10 % SDS and 3 µl Denhardt solution. Samples were heated for 3 min in a boiling water bath, cooled at room temperature for 5 min and centrifuged at maximum speed in a microfuge for 2 min to remove particulates. This mixture was put onto the microarray slide, sealed with a coverslip in a hybridization chamber and incubated for 18 h at 63 °C. Following hybridization, microarray slides were washed briefly in pre-warmed (60 °C) 1x SSC/0.03 % SDS to remove the coverslip and then washed twice for 5 min in each of the following buffers: a) 1x SSC/0.03 % SDS (pre-warmed to 60 °C), b) 0.2x SSC and finally c) 0.05x SSC. Microarray slides were dried by centrifugation at 300 x g for 15 min before scanning. Microarrays were scanned using an Axon 4000A scanner and images were acquired using GenePixPro 3.0 software (Axon). All microarray data were filtered to remove poor-quality data using four sequential cut-off values (Mark Reuter, IFR, personal communication). All data values were from features above 50 µm in diameter, with sum of medians above 50, regression coefficient squared values above 0.2 and with a sum of the signal : noise ratio greater than 3. A control experiment (comparison of two mRNA samples from replicated independent cultures of the wild-type strain) was used to estimate the boundaries between genes that were equally and differentially expressed in the two samples (Holmes et al., 2005). For the analyses described here, this boundary would detect changes equivalent to about 3.3-fold greater intensity in one of the fluorescence channels.