Controls (planktonic growth) did not contain A. castellanii. Cultures were incubated at 30 °C (growth temperature of A. castellanii) for 30 min, and then gentamicin was added to wells containing A. castellanii

to 100 μg μL−1 to eliminate extracellular bacteria (Alsam et al., 2006). After 2 h, A. castellanii cultures were centrifuged at 100 g for 5 min and resuspended in 1 mL of PYG712 broth containing 25 μg μL−1 of gentamicin to prevent the growth of extracellular bacteria. After an additional 2 hours, cultures were centrifuged at 10 000 g learn more for 30 s, pellets were resuspended in 1 mL of ice-cold RNA stop solution (19% ethanol, 0.1% sodium dodecyl sulfate (SDS), 1% acidic phenol) (Bernstein et al., 2002), and incubated on ice for 30 min. Following centrifugation at 10 000 g for 5 min at 0 °C, the RNA was immediately extracted from the pellets. For determination of survival of intracellular

E. coli O157:H7, cultures were generated in exactly the same way as cultures used for RNA extraction were selleck products generated. At the end of each time point, cultures were subjected to 0.1% SDS (final concentration) for 15 min to lyse A. castellanii and CFUs were determined. This level of SDS had no effect on the viability of E. coli O157:H7 grown planktonically (data not shown). RNA isolation, DNase treatment, subsequent purification, and determination of the absence of DNA was conducted as described previously (Carruthers & Minion, 2009). Samples were purified and concentrated using Millipore Microcon of YM-30 columns. RNA integrity and purity (absence of eukaryotic ribosomal peaks) were determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), with all samples measured having an Agilent RNA integrity number of 9.0 or higher and were void of detectable eukaryotic rRNA peaks (data not shown). Samples were determined to be free of contaminating genomic DNA by the absence of a product after 30 rounds of PCR. The microarray used for these studies has been described (Carruthers & Minion, 2009). It is based on PCR products representing 4756

genes printed to Corning UltraGAPS substrates. Target generation, labeling, reaction clean-up, hybridization, and pre- and posthybridization washes were all conducted as described previously using Cy3 and Cy5 dyes (Oneal et al., 2008; Carruthers & Minion, 2009). Scanning, image segmentation, and normalization were conducted as described previously (Oneal et al., 2008). Cluster of orthologous groups of proteins (COGs) information was obtained from NCBI (http://www.ncbi.nlm.nih.gov). Eighteen RNA samples, half from cells within A. castellanii and half from planktonic control cells, were used for the microarray study. A sample from each treatment was randomly paired with a sample from another treatment for hybridization on a two-color microarray substrate for a total of nine hybridizations.