Fast sg qpcr master mix

RNA isolation
and purification, reverse transcription, and qPCR were
performed according to previously published data.^{27 (link)} Briefly, RNA was isolated and purified from the S. aureus 5 N isolate (OD₆₀₀ = 0.5) after
samples were treated with sublethal doses of aPDI (reduction in bacterial
cell count ∼0.5 log₁₀ units) using a Syngen Blood/Cell
RNA Mini Kit (Syngen, Poland). The TranScriba kit (A&A Biotechnology,
Poland) was used to transcribe the RNA to complementary DNA (cDNA).
qPCR assays were performed using a LightCycler 480 II (Roche Life
Science, Germany). The reaction mixture (10 μL total) consisted
of 5 μL of Fast SG qPCR Master Mix (EURx, Poland), 200–400
nM of each primer (TIB MOLBIOL, Germany) (Table S1), nuclease-free water, and 1 μL of fivefold dilution
of cDNA. The following steps were implemented (Table S2). Melting curve analysis was carried out to exclude
primer-dimer formation or nonspecific amplification. Relative changes
in the expression of the sec, tst, srrA, and srrB genes were normalized
to the gmk reference gene.

Total RNA was isolated from cultured cells using a fenozol reagent (A&A Biotechnology, Gdynia, Poland). For the reverse transcription, 0.5 μg of total RNA was used and the reactions were performed according to the manufacturer’s instructions (EURx, Gdansk, Poland). Next, the RT-qPCR was performed with using a Fast SG qPCR Master Mix (EURx, Gdansk, Poland), and the gene expression was examined using a LightCycler 96 Instrument (Roche, Mannheim, Germany) under the following conditions: incubation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The expression of the target gene was normalized against glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The sequences of primers used for the RT-qPCR are presented in Table 1.