First strand cdna synthesis kit

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)].

Real-time quantitative Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of VEGFA, SP1 mRNA and miR-125b-5p. Total RNA (1 μg) was reversed to cDNA by using First Strand cDNA synthesis kit (Vazyme, Nanjing, China). A total of 20 μL mixed liquid, containing cDNA, primers and enzyme mixture was added to the PCR 96-well plates for PCR reaction in PCR instrument (ABI ViiA7, United States). The PCR reaction procedure was as follows: 95°C for 30 s, then 95°C for 10 s plus 60°C for 30 s for 40 cycles. GAPDH or U6 was used as endogenous control. Primers sequences were designed as follows:

VEGFA forward: 5′-TAG​AGT​ACA​TCT​TCA​AGC​CGT​C-3′,

VEGFA reverse: 5′-CTT​TCT​TTG​GTC​TGC​ATT​CAC​A-3′;

SP1 forward: 5′-CTG​GTG​GGC​AGT​ATG​TTG​TG-3′,

SP1 reverse: 5′-AAG​CTG​GCA​GAA​CTG​ATG​GT-3′;

GAPDH forward: 5ʹ-CTC​CTC​CTG​TTC​GAC​AGT​CAG​C-3ʹ,

GAPDH reverse: 5ʹ-CCC​AAT​ACG​ACC​AAA​TCC​GTT-3ʹ;

U6 forward: 5′-ATT​GGA​ACG​ATA​CAG​AGA​AGA​TT-3′,

U6 reverse: 5′-GGA​ACG​CTT​CAC​GAA​TTT​G-3′;

miR-125b-5p forward: 5′-ACT​GAT​AAA​TCC​CTG​AGA​CCC​TAA​C-3′,

miR-125b-5p reverse: 5′-TAT​GGT​TGT​TCT​GCT​CTC​TGT​CAC-3′.

Total RNA was isolated from pepper anthers according to the instructions of an RNA Isolation Kit (Huayueyang, Beijing, China). RNA was then reverse transcribed into cDNA using a first-strand cDNA Synthesis Kit (Vazyme). qRT–PCR was performed as described previously [24 (link)]. The primers are listed in Supplementary Data Table S1.