Transcriptional Analysis of Microbial Fermentation

Samples for real-time PCR (RT-PCR) analysis were collected in the late exponential growth phase during the fermentation experiment. The RNA in the samples was stabilized by adding two volumes of RNAprotect reagent (Qiagen Korea Ltd., Korea) and processed as dictated in the protocol. The cell pellets, as treated with RNAprotect, were stored at −80 °C prior to extraction of total RNA. A Nucleospin^® RNA isolation kit (MachereyNagel, Germany) was used to isolate the total RNA from the processed pellets. The RNA was quantified in a UV-spectrophotometer and checked in agarose gel for integrity and concentration. The isolated total RNA and random hexamers were used to synthesize cDNA using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific, Waltham, MA, USA). RT-PCR analysis was performed using the synthesized cDNA, gene-specific primers and Power SyBr^® Green (Thermo Fisher Scientific). RT-PCR was performed in a 48-well StepOne real-time PCR system (Thermo Fisher Scientific), and rpoD was used as an endogenous control. The experiment was performed in duplicate, and relative mRNA quantification was performed according to the ΔC_T method [35 (link)].