An overnight culture of Salmonella enterica serovar Enteritidis phage type 8 was diluted 1/1000 into 500 ml of LB at 37°C, then grown at 37°C for exactly 6h30min with vigorous shaking, at which point control samples were taken (OD approx. 0.5). Subsequently, trans-cinnamaldehyde (0.01% final concentration) or eugenol (0.04% final concentration) was added to the cultures, and 100ml samples taken 15min and 30min later. Cells were mixed with 16 ml ice-cold ethanol/phenol stop solution (5% citrate-buffered phenol), centrifuged and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Frozen cell pellets were resuspended in a fresh solution of 10ml 0.5 mg/ml lysozyme in TE (pH 8.0). A volume of 1ml of 10% SDS was added to the resuspended pellet, the solutions mixed and placed in a water bath at 64°C for 2min. Then, phenol/chloroform extraction was performed, followed by isopropanol precipitation. The pellet was resuspended in 2ml of RNase-free DEPC-treated water, split into two and DNAse treated by addition of 500U RNase inhibitor (Ribolock®, Fermentas), 250U DNase (Fermentas), 20µl of 1M Tris (pH 8.3) and 10µl of 1M MgCl, at 37°C for 30 min. The samples were again phenol-chloroform extracted and precipitated with isopropanol before final resuspension in DEPC-treated water.
Label
Cy5
Label protocol
50µg of RNA was combined with 2.4 µg random hexamers, denatured at 70°C, and quick-chilled in ice-water. The remaining components of the reverse transcription reaction were added to the denatured RNA and random hexamers to the following concentrations in a 60 µl reaction: 0.01 M DTT, 1X Superscript II first strand reaction buffer, 0.5 mM dATP, dCTP, dGTP, 0.2 mM dTTP, 0.066 mM Cy3- or Cy5-labeled dUTP (GE Healthcare), RNasin (Roche), and 800 U Superscript II. After 1 hour at 42°C, an additional 400 U of Superscript II was added and incubated another hour. Excess random hexamers and nucleotides were removed with a PCR Purification Kit (Qiagen) per manufacturer’s instructions.
An overnight culture of Salmonella enterica serovar Enteritidis phage type 8 was diluted 1/1000 into 500 ml of LB at 37°C, then grown at 37°C for exactly 6h30min with vigorous shaking, at which point control samples were taken (OD approx. 0.5). Subsequently, trans-cinnamaldehyde (0.01% final concentration) or eugenol (0.04% final concentration) was added to the cultures, and 100ml samples taken 15min and 30min later. Cells were mixed with 16 ml ice-cold ethanol/phenol stop solution (5% citrate-buffered phenol), centrifuged and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Frozen cell pellets were resuspended in a fresh solution of 10ml 0.5 mg/ml lysozyme in TE (pH 8.0). A volume of 1ml of 10% SDS was added to the resuspended pellet, the solutions mixed and placed in a water bath at 64°C for 2min. Then, phenol/chloroform extraction was performed, followed by isopropanol precipitation. The pellet was resuspended in 2ml of RNase-free DEPC-treated water, split into two and DNAse treated by addition of 500U RNase inhibitor (Ribolock®, Fermentas), 250U DNase (Fermentas), 20µl of 1M Tris (pH 8.3) and 10µl of 1M MgCl, at 37°C for 30 min. The samples were again phenol-chloroform extracted and precipitated with isopropanol before final resuspension in DEPC-treated water.
Label
Cy3
Label protocol
50µg of RNA was combined with 2.4 µg random hexamers, denatured at 70°C, and quick-chilled in ice-water. The remaining components of the reverse transcription reaction were added to the denatured RNA and random hexamers to the following concentrations in a 60 µl reaction: 0.01 M DTT, 1X Superscript II first strand reaction buffer, 0.5 mM dATP, dCTP, dGTP, 0.2 mM dTTP, 0.066 mM Cy3- or Cy5-labeled dUTP (GE Healthcare), RNasin (Roche), and 800 U Superscript II. After 1 hour at 42°C, an additional 400 U of Superscript II was added and incubated another hour. Excess random hexamers and nucleotides were removed with a PCR Purification Kit (Qiagen) per manufacturer’s instructions.
Hybridization protocol
Labelled cDNAs were hybridized to PCR product arrays on Corning UltraGAPS slides in 25% formamide / 5xSSC / 0.1% BSA hybridization buffer overnight at 42°C.
Scan protocol
The slides were scanned using the ScanArray 5000 laser scanner (GSI Lumonics).
Description
second replicate
Data processing
Image analysis and feature extraction was performed with Scanarray 2.1 software, and signals were quantified using Quantarray 3.0 (Packard BioScience).
