Quantitative Real-Time PCR for Gene Expression Analysis

Specific primer pairs were used to amplify total cDNA (30 ng) template (Table 1), using Maxima SYBR Green/Fluorescin qPCR Master Mix (2X, Thermo Scientific, Waltham, MA, USA). The thermal profile was: initial denaturation at 95°C for 5 minutes, then 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and final extension step for 5 minutes at 72°C. Samples were subjected to quantitative real-time PCR in duplicates, and then the mean values were used for subsequent analysis. Rotor-Gene Q MDx (Qiagen, USA) performed the amplification, automatically collected the data, and afterwards analyzed the value of threshold Cycle (Ct), which was normalized to an average Ct value of the housekeeping gene (∆Ct). The relative gene expression fold change was estimated using 2^−∆∆ct method,28 (link) standardized to the reference housekeeping gene 16S rRNA.Table 1

List of Primer Sequences Used for Quantitative Real-Time PCR (qRT-PCR)

Gene	Primer Direction	5′- 3′ Sequence	Reference
mecA	Forward	CCTAGTAAAGCTCCGGAA	[40 (link)]
	Reverse	CTAGTCCATTCGGTCCA
IcaA	Forward	TCTCTTGCAGGAGCAATCAA	[41 (link)]
	Reverse	TCAGGCACTAACATCCAGCA
IcaD	Forward	ATGGTCAAGCCCAGACAGAG
	Reverse	CGTGTTTTCAACATTTAATGCAA
16S rRNA	Forward	CGTGCCTAATACATGCAAGTC	[42 (link)]
	Reverse	CCGTCTTTCACTTTTGAACCA