RNA Isolation and Quantitative PCR

For RNA isolation, bacteria were grown in 20 ml of THY, harvested in the exponential growth phase (optical density at 600 nm = 0.3) and quickly frozen in liquid nitrogen. As described by Pappesch et al. [22 (link)], RNA was isolated with the Direct-zol RNA MiniPrep Kit (Zymo Research), subsequent acid phenol:chloroform:isoamyl alcohol (125:24:1) extraction and TURBO DNase treatment. cDNA was generated with the SuperScript first-strand synthesis system for RT-PCR (Thermo Fisher Scientific). SYBR green (Thermo Fisher Scientific)-based quantitative real time PCR was carried out on a ViiA Real-Time PCR System (Applied Biosystems). The 5S rRNA gene was used as a housekeeping gene. Primers used for S5nA and 5S cDNA detection are listed in Table 1.