Rna preparation kit

Each sample was pooled with five buds from the same walnut tree, and three biological repeats (from three different walnut trees) were performed for five different stages. The total RNA of 15 samples were extracted individually by using RNA preparation kit (Tiangen Biotech, Beijing, China) following the provided protocol. RNA concentration was measured using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Then, quantified RNA samples were used for constructing cDNA libraries subsequently.

Ag3GT, a key gene for anthocyanin synthesis, and AgFNS, a key gene for apigenin synthesis, were identified quantitatively (Tan, 2020 ). The expression levels of apigenin and anthocyanin-related genes were measured at 15, 30 and 45 days after different light intensities. Total RNA was extracted from the leaves and petioles of fresh celery by employing an RNA preparation kit (Tiangen, Beijing; 0.1 g). 1µg of total RNA was reverse transcribed to cDNA using TaKaRa reverse transcription kit, then diluted with sterile ddH₂O and stored in -20°C refrigerator and used for fluorescent quantitative PCR analysis. The fluorescent dye reagent is TB GreenTM Permix Ex TapTM II (TAKARA). Use the Archimed X Series real-time PCR instruments. The amplification program was as follows: 30 s at 95°C, 40 cycles of 5 s at 95°C, 15 s at 58°C. Relative gene expression was evaluated using the 2^-△△CT method. The PCR primers were designed using Primer Premier 5 software (PREMIER Biosoft, Palo Alto, CA, United States), and is listed in Table 1. Actin (AF111812) was used as an internal control.

The stigmas of different developmental stages in the S_13-bS_13-b line were set up with three biological replicates, 300–500 stigmas were collected for each replicate and placed in 1.5 mL of RNase-free EP tubes and immediately frozen in liquid nitrogen; the sample is stored at −80 °C. The total RNA was extracted from 9 samples of three stages of stigma development using the RNA preparation kit (Tiangen). The quality of purified RNA was initially evaluated on 1.5% agarose gel and then the RNA concentration was measured using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA).