Real master mix sybr green

Fifteen isolates of MDR strains of H. pylori were cultured on Mueller-Hinton agar, supplemented with 5% defibrinated sheep blood, 7% FCS, 1, 2, and 8 µg/mL of Cla, Amx, and Mtz, respectively. All plates were incubated at 37°C, under microaerophilic conditions. Bacterial suspension was prepared in normal saline. The cells were then isolated by centrifugation (9,000×g for 10 minutes), and the pellet was used for total RNA extraction. RNA in MDR strains was isolated using the RNA extraction kit (Hoffman-La Roche Ltd.) and then reverse transcribed into cDNA. The hefA vs gyrB (an endogenous gene) was used to study the relative expression of the hefA in 15 MDR clinical strains and strain ST7136 of H. pylori. cDNA of hefA and gyrB was amplified using RT-PCR (ABI StepOne™; Thermo Fisher Scientific, Waltham, MA, USA) in the presence of Real Master Mix (SYBR Green; TaKaRa, Shiga, Japan). The sequences of hefA (GenBank, accession number AF059041), including F: (5′-CTCGCTCGCATGATCGC-3 and R: (5′-CGTATTCGCTCAAA-TTCCCT-3′) were used to amplify a 162-bp fragment. The expression of the housekeeping gene (GenBank, accession number AB084049) was assessed in parallel with the primer pair gyrB: F: (5′-TTACTACGACTTATCCTGGGGCTAGCGCTG-3′) and R: (5′-CCCCATCAAT-TTCCACATTCTCCGC-3′), amplifying a 267-bp fragment. Then, the hefA expression in MDR and susceptible strain were determined.27 (link)