Quantifying mRNA Transcription Levels in MRSA

To analyze the mRNA transcription levels, qRT-PCR was performed as previously described [36 (link)]. MRSA DPS-1 suspensions (OD_600nm value of 0.7) were incubated with sub-inhibitory concentrations (1/2 × MIC, 1/4 × MIC, 1/8 × MIC and 1/16 × MIC) of SKN. After shaking the culture, the bacterial cultures were centrifuged at 13,000 rpm for 10 min to pellet the bacterial cells. Total RNA was extracted using the E.Z.N.A.^® bacterial RNA kit (OMEGA Bio-Tek, GA, USA) according to the manufacturer’s protocol. Equal mRNA amounts (1 μg) were determined by measuring the absorbance ratio at 260 nm and 280 nm using a NanoDrop spectrophotometer (Bio-Tek, Winooski, VT, USA). Then the mRNA was reverse-transcribed into the complementary DNA (cDNA) using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. 20 μL reaction mixtures containing SYBR master mix (Applied Biosystems, Massachusetts, USA), primers, sample cDNA, and deionized water were set up to run PCR using the StepOnePlus real-time PCR system (Applied Biosystems, France). The primer sequences used to synthesize the DNA template are listed in Table 2. The qRT-PCR results were normalized to 16S, the housekeeping gene for S. aureus.