Quantitative RT-PCR Analysis of CasR Gene Expression

The pMV261 empty and casR-overexpressing strains were grown to OD₆₀₀ of 1.0 in 100 mL of 7H9 medium. The extraction of mRNA from the cultures and qRT-PCR analysis was performed as described previously [23 (link)]. The cDNA was obtained using the HiScript II Q RT kit (Vazyme, Nanjing, China). The qRT-PCR system consisted of 20 μL of solution containing 10 μL of 2 × SYBR Green qPCR mix, 400 nM specific primers, and 1 μL of cDNA. The reactions were performed in a Bio-Rad CFX instrument under the following program: 95 °C for 1 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The expression level of each gene was normalized with sigA as an internal reference. Gene relative expression was determined according to the 2^−ΔΔCt method [28 (link)].