Qs6 real time pcr system

Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Along with genomic DNA (gDNA) removal, 500 ng of RNA was reverse transcribed using the TransScript All-in-One First-Strand cDNA Synthesis Kit (Transgenbiotech, Beijing, China). RT-PCR (SYBR) was performed using reagents from Transgenbiotech (Beijing, China) on a QS6 Real-Time PCR System (Applied Biosystems). Relative gene expression levels were calculated using the comparative threshold cycle (Ct) method and normalized to β-actin. The primers used for amplification are listed in Table 1.

Seven homolog efflux pump genes and the regulatory gene whiB7 (MAB_3508c) correlate with macrolides resistance in mycobacteria, were selected and analyzed. Relative expression of the genes was assessed by comparing the quantity of mRNA expressed by the organism cultured in the presence and absence of CLA using the same technical approach reported previously.28 A culture incubated in the presence of half its MIC of CLA was shaken at 37°C for 3 h; the RNA was then extracted according to the protocols described by Medjahed et al29 cDNA was synthesized using the HiScript III RT SuperMix with gDNA wiper (Vazyme Biotech Co., Ltd). qRT-PCR was performed using ChamQ Universal SYBR Master Mix (Vazyme Biotech Co., Ltd) on a QS6 Real-Time PCR System (Applied Biosystems, Carlsbad, CA). SigA was chosen as the endogenous reference gene. All PCR primer pairs used for amplification are shown in Supplementary Table 1. Calculation of fold change was described previously in detail.30 (link) Reactions were repeated in triplicate; genes with expression levels ≥4 were considered overexpressed