Nucreospin rna

Yeast cells harvested at 24 h of fermentation in YPD50, YPX50, and YPGN50 media were used for RNA preparation to observe the gene expression. The cells were homogenized using the Shake Master Neo (Bio Medical Science, Tokyo, Japan) and 0.5 mm glass beads. Total RNA was isolated from the homogenized cells using the NucreoSpin RNA (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA concentration and quality were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. cDNA was synthesized from 200 ng of total RNA using the RevarTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). Expression levels were quantified using a KOD SYBR qPCR Mix (TOYOBO) in the Mx3005P RT-PCR platform (Agilent Technologies). Amplifications were performed under the following conditions: 98 °C for 2 min; 40 cycles of 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s. A melting analysis was conducted at the end of the amplification cycle to verify the specificity of the reaction. Gene expression levels of target genes were normalized to that of the housekeeping gene TUB2. Primers used for qRT-PCR are listed in Additional file 2: Table S2.

Yeast harvested from the certain cultures were used for RNA preparation to observe the gene expression. Total RNA was extracted from the cells harvested at 48 h fermentation using the NucreoSpin RNA (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. The expression levels of ARO4 and ARO7 genes were quantified by real-time PCR as previously described (Inokuma et al., 2018 (link)). All the primers used for qRT-PCR are listed in Supplementary Table S1.