Sybr fast qpcr mix

Samples of bacterial culture were prepared as described above. Total RNA was extracted using the OMEGA bacterial RNA kit (OMEGA Biotech, Doraville, GA, USA) and eluted into 50 μl ddH₂O. Three microliters of RNA was reverse transcribed into cDNA using the TransScript TM Two-Step RT-PCR Super Mix (TransGene Biotech, Beijing, China) in a total volume of 20 μl. Using standard PCR and real-time PCR, 2 μl of cDNA was used to evaluate and represent the quantity of total mecA mRNA in each 2 ml sample because it held the same proportion of total RNA in each sample.
For standard PCR, 2 μl of cDNA was amplified via PCR with TransScript 2× PCR Super Mix (Transgene Biotech, Beijing, China) in a total volume of 30 μl. The primer pairs (mecAF/mecAR) are shown in Additional file 1: Table S1 [18 (link)].
For qRT-PCR analyses, real-time PCR amplification was performed in a total volume of 25 μl containing 12.5 μl of 2× SYBR Fast qPCR Mix (TAKARA, Dalian, China), 1.0 μl primer and 2 μl template cDNA. The specific primers (mecA-1501F, mecA-1598R) used for the detection of mecA gene are listed in Additional file 1: Table S1 [19 (link)]. Data were presented as the relative copies of mecA mRNA levels compared with that of untreated CoNS with a single SCCmec element.

Total RNA was isolated from liver tissue using TRIzol (Invitrogen). The cDNA was synthesized from the RNA samples. The real‐time quantitative polymerase chain reaction (qRT‐PCR) reaction was carried out using the SYBR^® Fast qPCR Mix (Code no. RR430A, TaKaRa, Japan) via the Real‐time PCR System (ABI Prism 7500, Life Technology, MA, USA). The qPCR reaction conditions were as follows: 95°C for 15 s, annealing at 60°C for 10 s, and extension at 72°C for 34 s for a total of 40 cycles. The primer sequences were shown in Supporting Information Table S3. GAPDH served as an internal control for all measurements. Each sample was amplified in triplicate. Relative gene expression data were analyzed using the 2^−ΔΔCT method (Sur et al., 2016).