Quantitative Real-Time PCR Analysis of Yeast Gene Expression

Total RNA was isolated using a yeast total RNA extraction kit (Sangon Biotech Co. Shanghai, China). Isolated RNA was treated with RNase-free DNase I (Toyobo, Osaka, Japan) and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan) as described (Wang et al., 2018 (link)). Real-time PCR was conducted on a Bio-Rad iCycler iQ (Bio-Rad, Hercules, CA, United States) using the THUNDERBIRD SYBR qPCR mix kit (Toyobo, Osaka, Japan). The primers for KmYME and the ACT1 internal control are shown in Additional File 1: Supplementary Table S1. The cycle threshold values (C_T) were determined and the relative fold differences were calculated using the 2^–ΔΔCT method (Nolan et al., 2006 (link)) with ACT1 as the endogenous reference gene. The fold change was shown as the sign of the log₂ transformed fold change (FC) values (log₂ FC).