RNA Extraction and Real-Time qPCR Analysis

Total RNA was extracted from the juice sacs according to the method described by Ma et al. (2022) [43 (link)]. The RNeasy Mini Kit (Qiagen, Germany) was used to clean the extracted total RNA using on-column DNase digestion. The cDNA was synthesized with 2 µg of purified total RNA using a TaqMan Reverse Transcription Regents (Applied Biosystems, USA).
The gene expression was conducted by real-time quantitative PCR according to the method of Ma et al. (2022) [42 (link)]. As an endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used. The real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) on a StepOnePlus™ system (Applied Biosystems). Each reaction contained template cDNA, 900 nM primers, and a 250 nM probe (Table S2). The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The data obtained from the StepOnePlus™ real-time PCR software (Applied Biosystems) was used to analyze the gene expression. The results were normalized with the results of 18 S ribosomal RNA. The Real-time quantitative RT-PCR was performed in three replicates for each sample.