Thunderbird next sybr

Total RNA was extracted from wild-type cells cultured in EMM + E or EMM + S medium in the presence or absence of SLF (3 mL culture). Cells were harvested by centrifugation, washed by ice-cold water, and suspended in 0.4 mL of ISOGEN (Nippon Gene), to which 0.3 g of glass beads were added. Cells were disrupted with Multi-Beads shocker (Yasui Kikai, 2000 rpm, 10 s). Total RNA was extracted according to the manufacturer’s protocol. The RNA concentration was quantified by a DU730 UV/vis spectrophotometer (Beckman Coulter). cDNA was prepared using the PrimeScript RT reagent kit (TaKaRa). qPCR was performed on Analytikjena qTOWER3G. THUNDERBIRD Next SYBR (TOYOBO) was used for RT-PCR. Primer sequences are listed in Table S5. The expression of tda1 was normalized to that of act1.

RNA samples were extracted using the RNeasy^® Mini kit (Qiagen) and reverse-transcribed to cDNA. Real-time quantitative PCR (RT-qPCR) was performed using THUNDERBIRD™ Next SYBR^® (TOYOBO, QPX-201), QuantStudio™ 3 Real-Time PCR System, and QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) with specifically designed primers (Supplementary Table S1). Data from three biological replicates were analyzed to calculate the relative fold change (2^−ΔΔCT). GraphPad Prism 9 software was used to plot the graphs.