Sybr fast qpcr master mix

Cells of the wild type strain XL280 and the indicated strains were cultured in YPD liquid medium at 30°C for 16 hr. Three biologically replicates were used. Total RNA samples were extracted using the PureLink® RNA Mini Kit (Life Technology) and their integrity was confirmed with RNA gel electrophoresis. Each RNA sample (8 μg) was treated with DNase (TURBO DNA-free™) according to the manufacture’s instruction. DNase-treated RNA samples were used as templates in the synthesis of the first strand cDNA using GoScript™ Reverse Transcription System (Promega) per the instruction from the manufacture. The resulting cDNA products were diluted to 8 ng/μl and 2 μl was used as template for quantitative real-time PCR (qRT-PCR). qRT-PCR was performed using the SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, MA) on a realplex² instrument (Eppendorf). The house-keeping gene TEF1 was used as the internal control for each sample to normalize the gene transcript level as we described previously (Chacko et al., 2015 (link)). The relative levels of transcripts were quantified using the ΔΔCt method as we described previously (Wang et al., 2014 (link); Wang et al., 2012 (link)). We used One-Way ANOVA for multiple comparison and P values ≤ .05 were considered statistically significant. Primers for qRT-PCR are included in the Supplemental Table 2.