Maximas sybr green fluorescein qpcr master mix

The total RNA was first collected using the Trizol reagent (#15596026, Life Technologies Corporation, Carlsbad, CA, USA), then 1 μg of total RNA was reverse transcribed into cDNA by using a QuantiTects Reverse Transcription Kit (#205311, Qiagen Sciences Inc., Germantown, MD, USA). C-DNA was amplified via a Maximas SYBR Green/Fluorescein qPCR Master Mix (#K0241, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) through specific primers that were prepared according to the manufacturer’s protocol (Table 1). For PCR assay, 12.5 μL Maxima SYBR Green/ Fluorescein qPCR Master Mix (2X) was mixed with 1 μL cDNA template, 0.3 μL forward primer, 0.3 μL reverse primer, and nuclease-free water to complete the volume to 25 μL. The conditions were designed as follows: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. β-Actin was applied as an internal reference for miRNA. Rotor-Gene^® Q with software version 2.1.0 (Qiagen Sciences Inc., Germantown, MD, USA) collected data automatically and analyzed the value of the threshold cycle (Ct), which normalized to an average Ct value of the house-keeping genes (∆Ct); 2^-ΔΔCt was used for calculating relative gene expression fold [26 (link)].

The influence of CDE on the expression levels of the biofilm genes (cna, fnbA, and icaA) was elucidated using qRT-PCR. After growing the isolates in TSB in the presence and absence of sub-MICs of SAM, they were incubated overnight at 37 °C. After the incubation period, cells were harvested by centrifugation and immediately stored at −80 °C. The total RNA from S. aureus isolates was extracted and purified using TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer protocol. Reverse transcription was employed using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Then, the formed cDNA was amplified using Maximas SYBR Green/Fluorescein qPCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). The average threshold cycle (CT) values were normalized to the housekeeping gene (16s rRNA). The relative gene expression of the treated isolates was compared to that of the untreated ones according to the 2^−∆∆Ct method [76 ]. Primers are exposed in Table S1 [77 (link),78 (link)].

According to the manufacturer’s procedure, the total RNA was purified from skin samples using TRIzols Reagent (15596026) (Life Technologies, Carlsbad, CA, USA). In a two-step RT-PCR experiment, 1 μg of total RNA was reverse-transcribed into single-stranded complementary DNA using the QuantiTects Reverse Transcription Kit (Qiagen, Hilden, FL, USA) and a random primer hexamer. QuantiTect Reverse Transcriptase is a hybrid of Omniscript and Sensiscript reverse transcriptases with a high affinity for RNA and the ability to produce cDNA from a wide range of RNA concentrations (10 pg to 1 μg). Unlike other kits, the QuantiTect Reverse Transcription Kit produces high quantities of cDNA templates for real-time PCR analysis independent of the location of the amplified target area on the transcript. C-DNA amplicons were amplified via Maximas SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) through specific primers (as shown by Table S4), which were prepared according to the manufacturer’s protocol.
Thermal cycling conditions were designed as follows: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. The conditions of the melting curve analysis were 72–95 °C, increased by 1 °C s⁻¹. Finally, the 2^−ΔΔCT method was performed to measure relative mRNA expression and normalized to β-actin [67 (link)].