Rnase free turbo dna free turbo kit

RNA isolation was performed as previously described [56 (link)]. Briefly, Leptospira spp. were grown until the growth reached exponential phase, OD₄₂₀ ~ 0.1 to 0.2 or ~ 2.5 × 10⁸ cells/mL. The cells were harvested and RNA was extracted using TRIZOL reagent (Thermo Fisher Scientific, Vantaa, Finland) as previously described [56 (link)]. RNA pellets were resuspended in UltraPure Dnase/Rnase Free Distilled Water (Thermo Fisher Scientific). Genomic DNA was removed by DNase treatment using the RNase-free Turbo DNA-free turbo kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The 500 ng of RNA were used for cDNA synthesis using iScript^™ Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad Laboratories, Hercules, CA). Quantitative reverse transcription-PCR (RT-qPCR) was performed using SYBR^® Green Master Mix (Bio-Rad). The results were expressed as the normalized difference of the threshold cycle (ΔΔCT), using cysK and lipL32 as a reference gene for L. biflexa and L. interrogans, respectively. All primers are listed in S1 Table.

Virulent L. interrogans serovar Manilae strain L495 and perR mutant M776 with less than three in vitro passages were used in this study. Four independent biological replicates of exponentially grown WT and perR mutant L. interrogans strains were incubated in the presence or absence of 10 μM H₂O₂ for 30 min at 30°C. WT L495 strain was also incubated in the presence of 1 mM H₂O₂ for 60 min at 30°C. Harvested cells were resuspended in 1 ml TRIzol (ThermoFisher Scientific) and stored at -80°C. Nucleic Acids were extracted with chloroform and precipitated with isopropanol as described elsewhere [44 ]. Contaminating genomic DNA was removed by DNAse treatment using the RNAse-free Turbo DNA-free turbo kit (ThermoFisher Scientific) as described by the manufacturer. The integrity of RNAs (RIN > 7.6) was verified by the Agilent Bioanalyzer RNA NanoChips (Agilent technologies, Wilmington, DE).