Cfx96 real time pcr instrument

Prior to the experimental operation, the bench was treated with a Solid RNase scavenger (Coolaber, Beijing, China). Frozen sweet potato storage root samples were ground in liquid nitrogen using a mortar and pestle to produce a powder from which total RNA was extracted using RNaiso Plus (Takara Biotechnology, Beijing, China). An ultra-low-volume spectrometer (BioDrop, Cambridge, UK) was utilized to measure the concentration and A260/A280 ratios for these samples, with cDNA then being prepared with a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology, Beijing, China), based on provided directions for RNA samples with an A260/A280 of 1.8–2.1. Prior to qPCR analyses, these cDNA samples were subject to 5-fold dilution. A Bio-Rad CFX96 Real-Time PCR instrument and TB Green^® Premix Ex Taq™ II (Takara Biotechnology, Dalian, China) were used for qPCR with the following settings: 95 °C for 30 s; 40 cycles of 95 °C for 5 s; 60 °C for 30 s. A melting curve from 60 °C to 95 °C (0.5 °C increments for 5 s) was used. The ∆∆Cq approach was used to assess relative gene expression [30 (link)] in the Bio-Rad Manager 3.1 software, with Actin serving as a control to normalize gene expression levels. Primer Premier 5.0 was used to design all primers for this study (Table S8).

Total RNA was isolated using the MiniBEST Universal RNA Extraction Kit (TaKaRa Bio, Code No. 9767) as specified by the manufacturer. The first-strand cDNA synthesis was carried out in accordance with the manufacturer’s instructions using a PrimeScript RT Master Mix (TaKaRa Bio, Code No. RR036A). The cDNA used for Real-Time Quantitative PCR analysis (qRT-PCR) was obtained by PrimeScriptTM RT Master Mix (TaKaRa Bio, Code No. RR036A). The qRT-PCR primer sequence is listed in Supplementary Table 2. The qRT-PCR experiments were carried out on a BioRad CFX96 real-time PCR instrument with TB Green Premix Ex Taq II (TaKaRa Bio, Code No. RR420A). The Chinese hickory ACTIN gene (CcActin) was used as a reference gene. All the expression analyses were repeated four times. Heat map was created by MeV software using average Ct values to show expression data.