Fecal bacterial isolation, DNA and RNA extraction was conducted as described previously (25 (link)). Levels of expression of E. coli adiA, cadA, and speA genes in the CRC and control microbiota cultures (from pooled total fecal bacteria, for each cohort) in response to acids, osmotic, and oxidative pressure were measured by quantitative reverse transcription (qRT)-PCR, with annealing temperature of 56°C for all reactions, as described (25 (link)). Mann-Whitney U tests for qRT-PCR (P < 0.05) were conducted to establish statistically significant differences in gene expression between the CRC and control groups. qRT-PCR was conducted using gene specific primers (Table 2 ), and 16S rRNA gene primers were used for normalization as described (25 (link)). Primer specificity was confirmed by Sanger sequencing by Eurofins after cloning PCR fragments into a TA pGEM-T Eazy cloning vector, Promega as described (25 (link)). Additionally, the SYBR Green iTaq (Bio-Rad) qRT-PCR system was tested with the 16S rRNA gene primers for contamination (water as the template) and DNA contamination of the RNA samples (proportionally to the amount of cDNA used for amplification diluted RNA samples were used as the templates for PCR). No amplification was observed for all control samples.
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