Quantifying Hormone-Induced Gene Expression in Kiwifruit

Total RNA was extracted from various kiwifruit samples (samples taken before and after each hormone/stress treatment) using the RNA Midi Kit (OMEGA, Guangzhou, China), following the manufacturer’s protocols. RNA extraction was conducted in triplicate using three biological replicates. Subsequently, reverse transcription was carried out using the First-Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). For quantitative real-time PCR (qRT-PCR), the Thermo Scientific Pikoreal Cycler 96 Real-Time PCR system was employed with SYBR Green Master Mix (Vazyme, Nanjing, China). The internal control for mRNA was AeActin1, and the relative expression levels of genes were determined using the 2^−∆∆CT method [41 (link)]. All experiments were performed in triplicate. Detailed information about the primer sequences can be found in Supplementary Materials Table S1. A heatmap was generated from real-time quantitative PCR analysis of AeIQM genes after hormone treatment. Values are the mean of three technical measurements. TBtools software (v2.027) normalized the data to obtain relative intensity values, with red indicating high expression levels and blue indicating low expression levels [38 (link)].