Analysis of gene expression
Gene expression was quantified using a quantitative PCR method. RNA from
frozen cells obtained from the previous step was extracted using a
NucleoSpin RNA® (Macherey-Nagel, Germany), purified
with rDNase to reduce DNA contamination and quantified using an Agilent
2100 Bioanalyzer (Mx3005P, Stratagene, Germany) in conjugation with the
RNA 600 Nano assay. Reverse transcription reactions were conducted to
convert the RNAs to cDNAs, which were subsequently quantified using an
Agilent Mx3005P qPCR. Primers used for quantification were specifically
designed for genes of interest in S. cerevisiae using the NCBI
Primer-BLAST Tool
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/)
with the target PCR product size set at approximately 100 bp, as listed
in Table S1. Relative gene expression was calculated using the
2-ΔΔCt method, and the TUB1 gene was
used as an internal reference gene.