Cfx96 qrt pcr system

GEFs were synchronized for 2 h with 100 nM dexamethasone (DXM, Sigma-Aldrich, St. Louis, MO, USA) in serum-free DMEM/F-12 medium containing 1 × AA. Cell samples were harvested at the indicated time points (after DXM synchronization for 24, 28, 32, 36, 40, or 44 h). Total RNA was extracted and purified from GEFs and goat liver and kidney tissues using TRIzol reagent (TaKaRa, Dalian, China) and then treated with RNase-free DNase (TianGen, Beijing, China). cDNA was generated using a PrimeScript RT Reagent Kit (TaKaRa). The primer sets used for the qPCR are listed in Table 1. The qPCR reactions were carried out in a 20 μL reaction solution comprising 10 ng cDNA with specific primers (200 nM) using the Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) and a CFX96™ qRT-PCR system (Bio-Rad, Hercules, CA, USA), as described previously (34 (link), 35 (link)). All reactions were performed in triplicate. The relative expression levels of each sample were normalized to the average level of the constitutively expressed housekeeping gene, RPLP0 (also known as 36B4).

qRT-PCR was used to verify the expression levels of the candidate DEGs. The total RNA was extracted from the 12 samples (three biological replicates of N1, B1, N2, and B2) and reverse-transcribed into cDNA for qRT-PCR. The ddH₂O was added in the qRT-PCR reaction mixture, including 2 μl cDNA, 2 µl BlazeTaq™ SYBR ^® Green qPCR mix2.0 (Applied Biosystems, Carlsbad, CA, USA), 2 μl qPCR Primer (2 μM), and 2 μl cDNA Template, to make up to 20 μl. The qRT-PCR analysis was performed by Bio-Rad CFX96 qRT-PCR system, and the reaction conditions were set as follows: 95°C for 15 min, PCR cycle step at 94°C for 20 s, annealing, and extension step at 60°C for 34 s. The specific primers were designed using Premier 5.0 software based on the Coding sequence (CDS) sequence of the target gene (Supplementary Table S3). The ACT7 in quinoa was used as the internal reference, and the calculation of relative expression of DEGs by the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) and t-test was applied to analyze the significant differences.

The quantitative real-time polymerase chain reaction (qRT-PCR) technique was used to verify the expression level of the differentially expressed genes (DEGs) related to the BETL development and starch synthesis process. The total RNA of the kernels from the WT and smk7a collected at 12 and 20 DAP was extracted using an RNA Easy Fast Extraction Kit (DP452, Tiangen, Beijing, China) and then transcribed into complementary deoxyribonucleic acid (cDNA) using qPCR RT Master Mix with gDNA Remover (KR116, TIANGEN, Beijing, China). The qRT-PCR reactions were carried out using Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme, Nanjing, China). The qRT-PCR analysis was performed using a Bio-Rad CFX96 qRT-PCR system. The reaction was carried out using three biological replicates with three technical replicates, with tubulin as the endogenous control (Zm00001d013367). The relative expression values were calculated using the 2^−ΔΔCt method [66 (link)], and the t-test was conducted to analyze the significant differences. The specific primers for the qRT-PCR are listed in Table S9.