Biosystems 7500 real time pcr system

Total RNA was isolated from kiwifruit samples and tobacco leaves according to the CTAB method^{53 (link)}. A NanoDrop 2000 instrument (NanoDrop Technologies, Inc., USA) was used to investigate the quality of the RNA samples. cDNA synthesis and qRT-PCR were performed as described by Wei et al.^{40 (link)}. qRT-PCR was conducted with a Biosystems 7500 real-time PCR system (Thermo Scientific, Inc., USA). Each primer pair without a template was used as a negative control. The 2^−ΔΔCt method was used to calculate kiwifruit and tobacco gene expression using AchnActin and Nbβ-Tubulin as internal standards. The analyses were conducted for three independent experiments. The primers used for qRT-PCR are presented in Table S1.

A total RNA extraction kit was used to extract RNA from cells after treatment. RNA concentration was measured with a spectrophotometer (NanoDrop ND-100, Shanghai Fishing Biotechnology Co., Ltd., Shanghai, China). PrimeScrip RT kit was used to reverse transcribe 1 μg of RNA into cDNA. With the applied Biosystems 7500 real-time PCR system (Thermo, Boston, USA), gene expression was quantified via quantitative real-time PCR reactions. Reactions were carried out in a total volume of 20 μL, including 10 μL SYBR Green mix, 2 μL cDNA, 0.4 μL Rox dye II, 6.8 μL H₂O and 0.4 μL forward and reverse primers. During the amplification process, the heat was initially applied at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s, and then gradually increased to 95 °C to obtain melting curve data. Primers were designed based on previous studies. The primer sequences used in this study were shown in Supplementary Table S1. As a housekeeping gene, β-actin was used to normalize the transcription of target genes. The relative gene expression was calculated using the 2^−∆∆Ct method.

The total RNA was isolated from Chinese cabbage tissues using a total RNA kit (Tiangen Biotech Co., Ltd., Beijing, China). The DNA-free total RNA (5 μg) was used for the first-strand cDNA synthesis in a 20 μl reaction volume (Thermo Fisher Scientific, MD, Lithuania) according to the manufacturer’s instructions. Real-time quantitative PCR (qRT-PCR) reactions were performed using the Bio-systems 7500 Real-Time PCR System (Applied Biosystems^®, Foster City, CA, United States) with the SYBR Green intercalating dye fluorescence detection. The amplification program was as follows: 3 min at 95°C, 40 cycles of 5 s at 95°C, 10 s at 55°C. Relative gene expression was evaluated using the 2^–ΔΔCt 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.