qRT-PCR was preformed SRT1720 mw on the same samples used for microarray analysis using primer sets for eight genes (dnaK, espA, lpfD, macA, ompA, recA, stx1A, stx2A) to confirm significant transcriptional differences due to treatment. The Express One-Step SYBR GreenER kit (Invitrogen) was used for qRT-PCR with the Mx3005P QPCR System (Stratagene, La Jolla, CA) and mxpro 4.1 software. Reaction volume for each well totaled 15 μL and
contained 3.69 μL of water, 7.5 μL qRT-PCR mix, 1.2 μL of each primer (Table 1) at 2.5 μM, 0.03 μL ROX, 1 μL of sample RNA, and 0.375 μL (75 U) SuperScript III. Six biological replicates for each treatment were randomly chosen for qRT-PCR validation and were run in duplicate. Gene btuD was used as a reference gene because it demonstrated no detectable differential expression due DAPT cost to treatment and had a
small variance on the microarrays. The method described in Gallup & Ackermann (2006) was used for primer optimization, detection of inhibition, and troubleshooting of qRT-PCR. A four-point standard curve was constructed with duplicate samples of a collection of all RNAs (Stock 1) and used for the calculation of efficiencies for target genes and the reference gene (Gallup & Ackermann, 2006). The ISU equation was used to calculate fold change between treatment and control samples (Gallup & Ackermann, 2006), and the Student’s t-test was used to determine significance of differences. Confidence threshold values that were greater than 2 SD from the mean were considered outliers and were Org 27569 not used in data analysis. The microarray dataset can be accessed from the National Center for Biological Informatics Gene Expression Omnibus using Series accession number GSE16762 (http://www.ncbi.nlm.nih.gov/geo/). Initially, we determined the survivability of E. coli O157:H7 in A. castellanii under the conditions of the microarray study (Fig. 1).
Initial CFUs of E. coli O157:H7 began at 109 and fell 5 logs during the first 2 h before leveling off to 103–104 for the next 14 h (Fig. 1). The addition of gentamicin to the culture media after a 30-min ingestion period did not affect the viability of A. castellanii or the bacteria within (data not shown). Microarrays were used to compare steady-state transcript levels of E. coli O157:H7 within A. castellanii to planktonic cultures to determine the effect of the intracellular environment. Based on the data from the internal survival curve, an incubation period of 4.5 h was chosen for the microarray study. This included an initial 30 min for A. castellanii engulfment of E. coli, 2 h for killing extracellular bacteria with gentamicin, and an additional 2 h for transcriptional activity to stabilize and allow dead bacteria to be degraded. All RNA preparations fulfilled our criteria for integrity and purity and lacked contamination with A.