RNA Extraction and Reverse Transcription

Strains were grown in 10 mL of LB broth at 37°C for 18–24 h up to the late exponential phase and collected by centrifugation. Total RNA extraction was carried out using the QIAGEN RNeasy purification kit. After checking the RNA extraction quality on a 1% agarose gel and measuring the RNA content (Nanodrop, Thermo Fisher Scientific, France), RNA extracts were stored at −20°C until further use. Prior to cDNA synthesis, genomic DNA (gDNA) was removed from 1 μg of total RNA using the gDNA wipeout buffer included in the QuantiTect Reverse Transcription kit (QIAGEN). The reverse transcription was performed in a volume of 20 μL including 14 μL of template RNA (extract concentrations adjusted to contain 1 μg of RNA), 1 μL of Reverse Transcription Master Mix, 4 μL of RT buffer 5× (containing dNTPs and Mg²⁺) and 1 μL of RT primer mix. Reverse transcription was performed in a Veriti PCR Thermal Cycler (Applied Biosystems, France) for 30 min at 42°C followed by a 3 min incubation at 95°C to inactivate the reverse transcriptase. All reactions including RNA handling were carried out on ice. The rpsL gene was used as reference to normalize the relative amount of mRNA.^{7 (link)}