Sybr green real time pcr master mix

Total RNA was extracted from treated cells with TRIzon reagent (CW Biotech, Beijing, China) according to the manufacturer’s instructions. cDNA was prepared from 100 ng of RNA using the HiFiScript cDNA Synthesis Kit (CW Biotech, Beijing, China). For miRNA and primary-miRNA reverse transcription, cDNA was prepared using a primer set from Genepharma (Shanghai, China). qRT-PCR analysis was performed by using a ViiA 7 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) and SYBR green real-time PCR Master Mix (CW Biotech, Beijing, China). Gene qRT-PCR primers are listed in Table S2. All qRT-PCR experiments were completed in triplicate.

The total RNA of each sample was isolated using the CTAB method (Yang et al., 2018 (link)) and digested with DNase (Takara, Dalian, China). Next, 0.5 μg RNA of each sample was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (CWBIO, Beijing, China). The cDNA was diluted 10-fold by sterile water and used as the template of qRT-PCR. The reaction mixture (20 μL) contained 10 μL of SYBR Green Real-time PCR Master Mix (CWBIO, Beijing, China), 0.5 μM of each forward and reverse primer, and 2 μL cDNA template (equivalent to 100 ng of total RNA). qRT-PCR was performed in StepOne™ Real-Time PCR System produced by Applied Biosystems. The amplification was achieved according to the following parameters: 94 °C for 30 s, followed by 44 cycles at 94 °C for 12 s, 60 °C for 30 s, 72 °C for 40 s, and at 81 °C for 1 s. The internal reference gene is walnut 18S rRNA (HE574850) (Xu et al., 2012 (link)), and the primers are shown in Table S1. The relative expression levels were calculated based on the threshold cycle using the 2^−ΔΔCT method (Livak & Schmittgen, 2001 (link)).

The total RNA of all samples were extracted by CTAB (cetyltrimethylammonium ammonium bromide) method [31 (link)]. The RNA concentration was determined and reverse-transcribed to cDNA by PrimeScript™ RT reagent Kit (CWBIO, Beijing, China) after treated by DNA digestion enzyme. The cDNA was diluted 10 times and used as the template of quantitative real-time PCR (qRT-PCR). QRT-PCR was performed using the SYBR Green Real time PCR Master mix (CWBIO) with an internal reference gene of walnut 18S rRNA (HE574850) [32 (link)]. The primers used are shown in Table S1. The instrument used for the quantitative reaction is the StepOne™ Real Time PCR system produced by Applied Biosystems. The reaction procedures were: 94 °C for 30 s, 45 cycles of 94 °C for 12 s, 60 °C for 45 s, 72 °C for 45 s; 81 °C for 1 s, 3 replicates per sample. The quantitative results were analyzed by 2^-ΔΔCT method [33 (link)]. The data were analyzed using the SPSS package (SPSS, Chicago, Illinois, USA). Sample variability is expressed as standard deviation. Expression differences between different time points and 0 d were analyzed by T test (P < 0.05). The results were visualized in Tbtools software and Origin 2017 and the results are represented using heat maps [30 (link)].

Isolated mRNAs were reverse transcribed by using SuperRTcDNA Kit (CWBIO, Beijing, China). Quantitative real-time PCR analyses on the mRNAs were performed by using the SYBR Green Real-time PCR Master Mix (CWBIO) in a Bio-Rad IQTM5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The thermal cycling program was set as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and annealing/elongation at 60 °C for 1 min. The mRNA levels of target genes were normalized to the β-actin mRNA levels. The primers used in this were synthesized by Invitrogen (Shanghai, China) and the sequences were showed in Supplementary Table S6.

Total RNA was extracted from leaf samples according to the instructions of Biofit kit (Tsingke, China). The strand cDNA was synthesized using Goldenstar RT6 cDNA synthesis kit (Tsingke, China). The expression levels of representative unigenes and selected key genes in the flavonoid biosynthesis pathway were analyzed by qRT-PCR using FQD-96A (Bioer Technology, China). The 20 μl reaction mixture contained 10 μl SYBR^® Green Real-time PCR Master Mix (CWBIO), 1 μl cDNA template, 0.8 μM of each forward and reverse primer, and 7.4 μl ddH₂O. The following cycling parameters were applied for amplification: 95°C for 1 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, then followed by 95°C for 5 s, 60°C for 1 min, 0.11°C / s to 95°C, 50°C for 30 s for plate reading. The primers list is shown in Supplementary Table S1. The actin gene was selected as an internal reference (Xu et al., 2019 (link)). Three replicates were performed for each sample. Quantitative data were analyzed using the 2^−ΔΔCT method.