One step qRT-PCR was completed from RNA with the qScript One-Step SYBR® Green qRT-PCR Kit (Quanta Biosciences), using a Bio-Rad iCycler CFX-96 machine. Primer design was aided by the Primer3 software available at http://www-genome.wi.mit.edu/genomesoftware/other/primer3.html (16 (link)). Unique primers were designed for 100–300 base pair regions of each gene in the urease gene cluster and a housekeeping gene (Table 2 ).
Standard PCR performed with genomic DNA from H. pylori strain G27 as the template was used to assure that all primer pairs resulted in amplification of a single product (data not shown). qPCR was completed using 10 QL reactions in a 96 well plate using the standard cycling protocol, conditions were 50o for 10 minutes, 95° for 5 minutes, followed by 45 cycles of 95o for 10 seconds, 60o for 30 seconds (data collection). Threshold cycle was calculated using the Bio-Rad software. A melting curve was used at the end of the run to confirm that there was only one peak and only one product for each primer. Results were analyzed using the comparative CT method (16 (link), 17 (link)) with gyrB used as a housekeeping gene. RNA isolation was completed two times for each condition, and each RNA sample was run in triplicate. Non-acidic pH (pH 7.4) was used as the denominator for comparison with acidic pHs (pH 3.0, 4.5, and 6.0).
Standard PCR performed with genomic DNA from H. pylori strain G27 as the template was used to assure that all primer pairs resulted in amplification of a single product (data not shown). qPCR was completed using 10 QL reactions in a 96 well plate using the standard cycling protocol, conditions were 50o for 10 minutes, 95° for 5 minutes, followed by 45 cycles of 95o for 10 seconds, 60o for 30 seconds (data collection). Threshold cycle was calculated using the Bio-Rad software. A melting curve was used at the end of the run to confirm that there was only one peak and only one product for each primer. Results were analyzed using the comparative CT method (16 (link), 17 (link)) with gyrB used as a housekeeping gene. RNA isolation was completed two times for each condition, and each RNA sample was run in triplicate. Non-acidic pH (pH 7.4) was used as the denominator for comparison with acidic pHs (pH 3.0, 4.5, and 6.0).
