Sybr premix ex taqtm tli rhaseh plus reagent

Total RNA was extracted and purified from cells grown on GYM medium for the indicated time using RNAiso Plus (TaKaRa) according to the manufacturer's instructions. One μg of each of the total RNA was used as a template for reverse transcription (RT), which was carried out using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). Realtime quantitative PCR (qRT-PCR) was carried out using an Applied Biosystems StepOnePlus Real-Time PCR System with SYBR^®Premix Ex Taq^TM (Tli RHaseH Plus) reagent (TaKaRa, Dalian, China). The gene primers used in qRT-PCR reactions are listed in S1 Table. Each reaction mixture was comprised of 7.5 μL of SYBR^®Premix Ex Taq^TM (2×), 1 μL of template, 0.3 μL of forward primer, 0.3 μL of reverse primer, 0.3 μL of ROX Reference Dye (50×), and 5.6 μL of RNase-free H₂O. The acceptable qRT-PCR standard curve (0.9≤E≤1.0, R²≥0.99) for each gene examined in this study was optimized by altering the annealing temperature and time. For each gene, all PCR reactions were carried out in triplicate within a single plate, with hrdB of S. diastatochromogenes 1628 used as the reference gene. Quantification of relative gene expression was analyzed using the 2^-ΔΔCt method[14 (link)].

Total RNA of T. brevicompactum 0248 and Δtri11, Δtri3, and Δtri4 knockout strains were isolated using Spin Column Fungal Total RNA Purification Kit (Sangon, Shanghai, China). The total cDNA was obtained through reverse-transcription reaction using PrimeScript^® RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). QRT-PCR was performed in an Applied Biosystems StepOnePlus Real-Time PCR System using a SYBR^® Premix Ex TaqTM (TliRHaseH Plus) reagent (TaKaRa, Dalian, China) (Shentu et al. 2014a (link)). The primers used in qRT-PCR are listed in Additional file 2: Table S2. The acceptable qRT-PCR standard curve (0.95 ≤ E ≤ 1.05, R² ≥ 0.99) of the gene examined in this study was optimized by varying the annealing temperature and annealing time. The β-tubulin gene of T. brevicompactum 0248 was used as the reference gene (Shentu et al. 2014a (link)). Quantification of the relative gene expression was analyzed by the 2^−ΔΔCt method.