Analyzing Mycobacterial Gene Expression via Real-Time PCR

Isolation of mRNA and cDNA from mycobacterial strains was performed as described previously33 (link). For real-time PCR analysis, gene-specific primers (Table S4) were used, and first-strand cDNAs were synthesized using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Each PCR reaction (20 ml) contained 10 ml of 2× SYBR Green Master Mix Reagent (Applied Biosystems), 1.0 ml of cDNA samples, and 200 nM gene-specific primers. The reactions were performed in a Bio-Rad IQ5 RT-PCR machine. The thermocycling conditions were 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. Amplification specificity was assessed by conducting melting curve analysis. Differential gene expression was normalized to the levels of 16S rRNA gene transcripts. The degrees of expression change were calculated using the 2^−ΔΔCt method34 .