Dntp mixture

Total RNA was extracted using the acid guanidium thiocyante/phenochloroform method, using a total RNA isolation reagent (Trizol Reagent, Invitrogen,USA) according to the manufacturer’s instructions. The RNAs were reverse-transcribed to cDNAs in a Thermal Cycler using Oligo (dT) primer (Invtirogen), RNase Inhibitor (Takara), Reverse transcriptase (Takara), dNTP Mixture and RNA PCR buffer (Sigma). A quantitative RT-PCR assay was carried out with a LightCycler™ (Roche Diagnostics, Mannheim, Germany) using the double-stranded DNA dye SYBR Green 1 (Roche Diagnostics) in order to observe the level of mRNAs. Primer sequence and size used in this study were shown in Table 2. Quantification was performed by comparing the levels obtained with standard samples as a previous study [21 (link)]. In the present study, the concentrations of cDNA in the unstimulated samples were 0.2, 0.5, 1.0 and 2.0 μl. Melting curve analyses were performed after the PCR amplification and to confirm no primer dimmer in the PCR products. The ratios of HSP70 mRNA expression were adjusted by the value of the housekeeping gene GAPDH.Table 2

Primer sequence and size used in this study

Gene	Sequence		Size
HSP70	Forward	5’-CGGACGAGTACAAGGTTGA-3’	206
	Reverse	5’- CTCTTTCTCCGCCAACTG-3’
GAPDH	Forward	5’-AGGGGCTCTCCAGAACATCA-3’	196
	Reverse	5’-GCCTGCTTCACCACCTTCTT-3’

The detection of genes encoding the efflux pumps (AdeB, AdeG, AdeJ, AdeY, and AbeM) and porins (oprD and carO) was investigated by PCR. Primers are listed in Supplementary Table S1. PCRs were performed containing 2.5 mM dNTP mixture (Sigma Aldrich), 1 unit (0.2 µL) of Taq Polymerase (Sigma Aldrich), 10 pmol of each primer, and 1 µL of bacterial DNA in a final reaction volume of 25 µL. The following parameters were used for thermal cycling: DNA denaturation at 95 °C for 5 min, 35 cycles of 95 °C for 30 s, amplification at 55–60 °C for 30 s, extension at 72 °C for 1 min, and final elongation at 72 °C for 5 min. PCR products were run on agarose gel electrophoresis, stained with EtBr (ethidium bromide), and gel images were captured after visualization on a UV transilluminator.

When H. pylori strains were successfully cultured from both the antrum and body in some patients, Randomly Amplified Polymorphic DNA (RAPD) fingerprinting was performed to confirm whether they were different strains. PCR-based RAPD fingerprinting was performed using the method as previously described [31 (link),32 (link)]. The primers (Cosmogenetech, Seoul, Korea) used were 5’-AACGCGCAAC-3’ (3881) and 5’-AAGAGCCCGT-3’ (3880). The Ex Taq™ DNA polymerase (Takara Bio, Otsu, Shiga, Japan) was used for PCR amplification, which was performed in a volume of 50 μL, containing 5 μL 10 × Ex Taq™ buffer including MgCl₂, 4 μL dNTP mixture (2.5 mM each; Sigma Chemical Co., St Louis, MO, USA), 10 pmole of each primer, and 1.25U Ex Taq™ DNA polymerase, following previously described methods [31 (link),32 (link)]. A GeneAmp^® PCR system 2700 (Applied Biosystems, Foster City, CA, USA) was used for amplification. The PCR profile consisted of 4 cycles of 5 min of denaturation at 94 °C, 5 min of annealing at 36 °C, and 5 min of extension at 72 °C; 30 cycles of 1 min of denaturation at 94 °C, 1 min of annealing at 36 °C, and 2 min of extension at 72 °C; and then 10 min of extension at 72 °C. After PCR-based RAPD fingerprinting, the products were electrophoresed in 1.0% agarose gel (Sigma Chemical Co., St Louis, MO, USA) and photographed under UV light.