Real-time quantitative reverse transcription PCR (qRT-PCR)
Cells used for RNA extraction were cultivated in 30 mL liquid LB medium at 37°C and harvested in the exponential growth phase. Total RNA ofE. coli was extracted using the High Pure RNA Isolation Kit (Roche Diagnostics, Basel, Switzerland) according to manufacturer’s instructions. Complementary DNAs (cDNAs) were synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, MA). Sequence specific primers were used for reverse transcription ofhemB mRNA (i.e., q-hemB) and internal control rrsA(encoding ribosomal RNA 16S) mRNA (i.e., q-rrsA) (Table 1), at a final concentration of 1 µM. 100 ng of the total RNA was used in a 20 µL reaction mixture. Real-time qRT-PCR was carried out using the Power SYBR® Green PCR Master Mix (ThermoFisher Scientific; MA) in an Applied Biosystems StepOnePlus™ System as per the manufacturer’s instructions. All experiments were performed in duplicate.