Mesa green qpcr mastermix plus for sybr

Quantitative real-time PCR (qRT-PCR) was performed with MESA GREEN qPCR MasterMix Plus for SYBR (Eurogentec) using CFX Connect Real-Time PCR system (Bio-Rad). The ΔakuA strain and mutants were grown in liquid MM medium for 20 h at 37°C and RNA was extracted using RNeasy Plant Mini Kit (Qiagen). DNase digestion and subsequent cDNA synthesis was carried out using 0.8 μg of RNA with the QuantiTect Reverse Transcription Kit (Qiagen). To perform the shift experiments, the indicated strains grown on liquied MM medium for 18 h at 37°C. Afterwards, the mycelium were shifted to liquid MM medium for 8 h or solid MM plate for 24 h. Addition of Doxycycline (5 mg/L) was noted. Each sample was performed in duplicates and the experiment was repeated two times. The histone H2A (3G05360) was used as reference gene for normalization. The relative expression of the gene of interest was calculated using the ΔΔCT method as previously described [91 (link)]. All the primers used for quantitative real-time PCR were listed in S3 Table.

Total RNA from cells were extracted using TRIZOL reagent (Invitrogen) following the manufacturer's instructions. Reverse transcription was performed with 0.5–1 µg of total RNA using Superscript III (Invitrogen), following the manufacturer's instructions. Real-Time PCR (RT-PCR) was carried in duplicates with 2 µg of transcribed cDNA and MESA Green qPCR MasterMix Plus FOR SYBR (Eurogentec) in a LightCycler 480 II sequence detection system (Roche Applied Science). PCR products were verified through dissociation curve analysis using SDS software (Roche Applied Science). Expression levels were normalized to TATA box-binding protein 1 (Tbp1) mRNA and represented in arbitrary units. The sets of primers used in these experiments can be seen in Table 1.