Transstart sybr green qpcr supermix udg kit

After cDNA synthesis, qPCR was performed to determine the expression levels of biofilm‐ and secretion‐related genes hcp1, hcp2, hcp3, lasR, pslA, pelA, pilA, and fliC using the TransStart SYBR Green qPCR SuperMix UDG Kit (TransGen Biotech) and the Eppendorf Real‐Time system (Eppendorf). Primers (Sangon Biotech) used for qPCR are listed in Table 1. A control lacking the template was included in the analysis as a negative control. For quantification, 16S rRNA was used as a marker gene. Relative expression levels were determined using the 2^−ΔΔCt method. Reactions were prepared according to the manufacturer's instructions with 10‐fold dilution of cDNA as the template. All reactions were carried out in triplicates with at least two biological replicates.

Three different efflux pump‐encoding genes, adeB, abeM, and amvA, were analyzed by quantitative reverse transcriptase–PCR (qRT‐PCR). The primers used for qRT‐PCR analysis are listed in Table 1. Gene expression was determined by qPCR using the TransStart SYBR Green qPCR SuperMix UDG Kit (TransGen Biotech). A no‐template control was included in the analysis. RpoB was used as the reference gene. Relative expression was determined using the 2^−ΔΔCt method with the Eppendorf Real‐Time System. Reaction samples were prepared according to the manufacturer's instructions with a 10‐fold dilution of cDNA. All reactions were carried out in triplicate with at least two biological replicates. Target gene expression was measured as the relative expression compared to that of rpoB.