Chamq universal sybr qpcr master premix

The total RNA of yeast strains cultivated in three M3 media for 12 h was extracted using the RNA simple Total RNA kit (Tiangen Biotech (Beijing) Co., Ltd.), following the manufacturer’s instructions. Subsequently, DNA elimination and reverse transcription were performed using the HiScript II QRT SuperMix (Vazyme Biotech Co., Ltd., Nanjing, China). Then, PCR was quantified on the CFX96TM RT-PCR system (Bio-Rad, Hercules, CA, USA) after adding the ChamQ Universal SYBR qPCR Master Premix (Vazyme Biotech Co., Ltd.). Finally, the relative expression levels of mRNA were determined using the 2^−∆∆CT method [44 (link)]. Among them, ΔCTs were derived by the CTs (cycle thresholds) of the target genes (YAT, GOT1, his C, PDC, and ADH5) minus the CT of ENO1, which served as the housekeeping gene. ΔΔCTs were calculated by ΔCTs from the target genes minus the CT of the control gene. Fold changes were determined using the 2^−∆∆CT method. The primers of related genes are listed in Table 2.

The yeast was cultivated to log phase and harvested. Total RNA was extracted using a Total RNA kit (TIANGEN, China). cDNA was generated using HiScript II QRT SuperMix with gDNA (Vazyme Biotech, China). After that, a quantitative analysis of the mRNA was performed by using a ChamQ Universal SYBR qPCR Master Premix (Vazyme Biotech, China) on a CFX96TM Real-Time System (Bio-Rad, USA). In this study, the ENO1 gene was used as a loading control for normalization. The relative changes in the mRNA level were calculated using the 2^−∆∆CT method, where CT is the threshold cycle. The primers used are listed in Additional file 1: Table S2.