Nanodrop spectrophotometer

Total RNA from the leaves was extracted with a total RNA isolation kit (Denazist Asia, Mashhad, Iran) based on the manufacturer’s instructions. The quantity and quality of the extracted RNA were evaluated on 1% agarose gel electrophoresis and by a NanoDrop spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). About 0.4 μg of total extracted RNA was used for cDNA synthesis.

RNA was isolated from the tissue or cells using the TRIzol reagent (Invitrogen, Waltham, MA, USA) and concentration as well as purity (^A260/^A280 ratio) was measured using a Nanodrop spectrophotometer (BioTEK, Winooski, VT, USA).cDNA was generated from RNA the Verso cDNA synthesis kit (ThermoFisherScientific) and cDNA was stored at − 20 °C. Realtime PCR assays were performed using a SYBR green master mix (Bio-Rad, Hercules, CA, USA) and QuantStudio3 thermocycler (ThermoFisher Scientific). Data was analyzed using the comparative C_T2^−ΔΔC_T method. All values were normalized against a calibrator gene (U6 for miRNA-21 and GAPDH for all others) and expressed relative to gene expression in control samples. Synthesis of Pre-rRNA in samples was determined by previously published protocol [13 (link)]. A complete list of the primers used is included in Additional file 1: Table S5.

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.