Mastercycler ep

RNA was isolated using TRIzol (Invitrogen) and transcribed into cDNA (Omniscript, Qiagen, Hilden, Germany) using random hexamer primers after DNAse digestion (Promega, Mannheim, Germany). cDNA of interest was amplified using primers listed in Supplementary Table 1 and 2 × DyNAmo Color Flash SYBR Green Master Mix (Thermo Scientific, Vienna, Austria) in an Eppendorf Mastercycler ep realplex^{2 (link)} cycler. Relative expression of target/housekeeper was calculated using the delta-CT method.

Total RNA was separated through total RNA extraction TRIzol reagent (Invitrogen Life Technologies) and centrifuge columns. The progress was conducted in the Mastercyclerep realplex PCR system (Eppendorf, Hauppauge, NY). Primer sequences for qRT‒PCR are listed in Supplementary Table 1.

Total RNA was extracted from THP-1 using QIAzol reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The RNA concentration was determined by measuring the samples’ absorbance at 260 nm by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA), and its purity was assessed by the absorbance ratio 260/280 nm and 260/230 nm. For each sample, 1 µg of RNA was reverse transcribed into complementary DNA (cDNA) using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Subsequently, Real-Time PCR was performed using the GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) to evaluate the gene expression of HO-1 and RPS18, used as reference gene. All PCR reactions were performed in triplicate using the Mastercycler ep (Eppendorf, Hamburg, Germany) with the following conditions: initially, 2 min of incubation at 95 °C, followed by 40 cycles consisting of 30 s at 95 °C, then 60 °C for 1 min and 30 s at 68 °C. The gene expression analysis was carried out according to ΔΔCt method [44 (link)].

Each PCR was performed in an Eppendorf Mastercycler ep gradient thermal cycler with 2 µl 10× PCR buffer; 2 µl MgCl₂ (50 mM); 1.2 µl of a mix of deoxynucleoside triphosphates (dNTP) (2 mM); 0.5 µl (each) primer (10 µM); 0.2 µl of Taq polymerase (Invitrogen); and either 1 µl of DNA or traces of cells and water to reach a volume of 20 µl. The following conditions were used: initial denaturation at 94°C for 3 min; 30 cycles with denaturation at 94°C for 40 s, annealing at 55 or 60°C for the MTL locus for 40 s, and extension at 72°C for 1 min/kb; and a final extension time at 72°C for 10 min. The PCR products were verified by electrophoresis on a 1% or 2% agarose gel.

Confirmation of H. pylori strains was performed using oligonucleotides 16S1 and 16S2 (Table 1), which amplify a 522 bp fragment of the 16S rRNA, according to the method described by Román-Román et al. [25 (link)]. vacA genotyping and the status of cagA and babA2 were assessed by PCR with oligonucleotides specific for each region and gene (Table 1). The reaction mixture contained 1.5 mM MgCl₂; 0.2 mM dNTPs; 2.5 pmol of oligonucleotides F1 and B1, or 5 pmol of VAGF and VAGR, or 2.5 pmol of VAIF and VAIR, or 12.5 pmol of babA2F and babA2R; 1.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and 200 ng of DNA, in a total volume of 25 µl. Amplification conditions were: one cycle at 94 °C for 10 min; 35 cycles at 94 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min; and a final extension cycle at 72 °C for 10 min. The PCR products were subjected to 2.5 % agarose gel electrophoresis, stained with ethidium bromide and visualized with ultraviolet light (UV). In each PCR, DNA from strain ATCC 43504 (vacAs1m1/cagA⁺/babA2⁺) was used as a positive control, and as a negative control, DNA was replaced with sterile deionized water. All reactions were performed in a Mastercycler Ep gradient thermal cycler (Eppendorf, Hamburg, Germany).