Turbo dnase enzyme turbo dna free kit

The RNA from Mtb strains was isolated as described in Sharma et al. (2019 (link)). Approximately 2 × 10⁹ WT and KO mycobacterial cells were used for RNA extraction. The RNA was isolated from the WT and KO pellets with a DNA, RNA, and protein purification kit (Machery-Nagel NucleoSpin^TM; Germany) as per the manufacturer's protocol. Three μg of the eluted RNA were treated with 1 μl of Turbo DNase enzyme (Turbo DNA-free Kit; Thermo Fischer Scientific, United States) to avoid contaminating the genomic DNA. Two μg of the DNase-treated RNA was used as template to generate cDNA as per PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). One μl of the generated cDNA for each sample was taken for quantitative real-time PCR (qRT-PCR) using 5 × HOT FIREPol Evagreen qPCR Mix Plus (SYBR Green; Solis Biodyne, Estonia) on the Stratagene mx3005p system (Agilent Technologies, United States). The primer pairs used are indicated in Supplementary Table 3. sigA transcript levels (Ct value) in different strains were used as internal controls for normalization and accurate estimation of Ct values of all genes under study.

Total RNA was extracted using a mirVana microRNA Isolation Kit (Thermo Fisher Scientific). All RNA samples were then treated with TURBO DNase enzyme (TURBO DNA-free Kit; Thermo Fisher Scientific). To detect mRNAs, cDNA was synthesized with a PrimeScript RT Master Mix (TaKaRa Bio) in accordance with the manufacturer’s instructions. Quantitative real-time RT-PCR (qRT-PCR) was performed using a SYBR Select Master Mix (Thermo Fisher Scientific). GAPDH mRNA was used as an internal control. The primer pairs used are listed in Supplementary Table S2. qRT-PCRs were performed in triplicate using a StepOne Plus real-time PCR system (Thermo Fisher Scientific).