qRT-PCR of ex vivo Salmonella enterica in NRAMP-1 minus/plus macrophage background

A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the NRAMP-1 (natural resistance-associated macrophage protein-1) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a NRAMP-1 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains ( WT and TMS) grown under 2 conditions (NRAMP-1 wildtype (plus) or NRAMP-1 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in duplicates, located on the same PCR plate. For each duplicate the mean value was calculated and used as a data point for further normalization. We averaged the Cp values for all the data points in a sample and then subtracted this sample average Cp from all the data points of the sample. Only 192 primer pairs from 288 on the platform GPL11111 were used.