Realplex2

Total RNA (4 µg) was treated with DNase I (Sigma), reverse transcribed with Revertaid Reverse Transcriptase (Fermentas) in a 25 µl reaction volume and amplified with Mastercycler ep Realplex (Eppendorf) using 5 µl of 30-fold diluted cDNA solution. Primers were designed with primer3 software and their respective sequences are shown in Table 1. Real-time PCRs were performed with the MaximaTM SYBR Green qPCR Master Mix (Fermentas) and 0.23 mM of each primer (Fermentas) in a 15 µl reaction. Cycle thresholds (Cts) were calculated using the Realplex 2.0 software (Eppendorf). For each plate and each gene, a standard curve made with dilutions of cDNA pools was used to calculate the reaction efficiencies, and the relative expression was calculated according to Hellemans et al. [20] (link) with HaEF1, Haβ-tubulin [12] (link) and HaS19 as reference genes (Table 1). An arbitrary value of 1 was assigned to dormant seed samples, which were used as control samples for normalization [21] (link). Results presented are the means ± SD of three biological replicates.

Six E. coli O157:H7 strains were selected from feedlot steers that had originally been sampled in California and Texas (from objective 1). Three strains were defined as normal shedders, isolated from cattle with concentrations of <10⁴ MPN/g of feces and three were defined as super-shedders, isolated from cattle with concentrations of ≥10⁴ MPN/g of feces. Each selected strain was also confirmed by Real-time PCR (Realplex 2.2; Eppendorf, Hauppauge, New York, USA) to contain at least one or both Shiga toxin genes (Philpott & Ebel, 2003 , Chapters 1 and 4).

Since some target DNA copies serving as templates for a PCR may be damaged or fragmented (e.g., during extraction and freeze-thaw during handling), DNA mass measurements may overestimate copy numbers [29 ]. Hence, the term “PCR forming units” (PFU) was introduced to clearly distinguish between copies being truly amplified during PCR and crude copy number estimates based on the measured DNA amount [30 (link)]. Based on this framework, the number of PFU per standard was calculated following the SIMQUANT assay [29 ]. SIMQUANT assumes a ratio of positive:negative PCR reactions of 7:3 (70% detection) during a series of PCR reactions with approximately 1 PFU. Accordingly, the standard containing 1 PFU was roughly identified and the DNA contents of the remaining standards were successively converted into respective PFU. These values were used to generate a calibration curve, where the mean Ct value for each standard (n = 7) was plotted as a function of the corresponding PFU. Subsequently, the LoD and LoQ were derived following the descriptions in Vrålstad et al. [28 (link)]. For a full description of the calculations for the LoD and LoQ, see S3 File. The software Real Plex 2.2 (Eppendorf, Hamburg, Germany) and R for Mac version 3.0.2 [31 ] were used for data analysis and preparation of figures.

To detect the expression of IL-2 receptor and GM-CSF receptor, total RNA was extracted using Trizol (Life technologies, Carlsbad, CA) and reversely transcribed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Life Sciences, Ontario, Canada) after DNase I (Fermentas) treatment. The sequence of primers used were as below: for mIL-2Rα, FP: 5′-GCAACTCCCATGACAAATCG-3′, RP: 5′-CCCGGAATACACTCGTAGTGAA-3′; for mGM-CSFRα, FP: 5′-CGTGCATATCCCCACCGTAATA-3′, RP: 5′-TGAAGGCACGTTGGATTTTATGA-3′; for GAPDH, FP: 5′-GCACGGTCAAGGCTGAGAAC-3′, RP: 5′-GCCTTCTCCATGGTGGTGAA-3′. Real-time quantitative PCR (qRT-PCR) were performed using the iQ™ SYBR^® Green Supermix kit (Bio-Rad, Hercules, CA) on Mastercycler ep realplex⁴ (Eppendorf, Hamburg, Germany). Target mRNA quantification was analyzed using the comparative threshold cycle (Ct) method with the software realplex 2.2 (Eppendorf) as described previously [36 (link)].

Total RNA was isolated from yeast cells by the hot phenol method85 . RNA (10 ng) was used with One Step SYBR^® Prime Script^TM Kit II (Perfect Real Time) kit according to the manufacturer’s protocol. The realplex 2.2 software (Eppendorf) was used to analyse the data and to calculate cycle threshold (C_t) values. Actin1 was taken as housekeeping gene. The change in gene expression was calculated as relative fold change by the comparative C_t method86 (link).

RNA isolation from cells was performed using Trizol reagent (Invitrogen) and c-DNA was synthesized by Improm II reverse transcriptase system (Promega). The absorbance at 260 and 280 nm was measured using NanoDrop ND-1000 UV-Visible Spectrophotometer. A260/A280 ratio of 1.9 to 2.1 indicated good quality of RNA and c-DNA.
Quantitative real time PCR was performed using SYBR Green Supermix (Biorad) in Realplex Real-Time Thermal Cycler (Eppendorf). The profile of thermal cycling consisted of initial denaturation at 95 °C for 2 min, and 40 cycles at 95 °C for 15 s and 60 °C for 45 s for primer annealing and extension. Melting curve analysis was used to determine the specific PCR products. The changes in the threshold cycle (C_T) values were calculated by the equation: - ∆C_T = C_{T (target gene)} - C_{T (endogenous control gene)} and fold difference was calculated as . C_T values and melting curves were analyzed on Eppendorf Realplex 2.2 software. GAPDH was used as an internal control to normalize gene expression. Fold change for each treated sample was calculated in comparison with constitutive m-RNA levels of the specific gene and graphs were plotted. Sequences of primers used in the study are listed in the Supplemental Table 1.