*2.3. RNA Extraction, cDNA Synthesis, and Gene Expression Analysis by Quantitative Real-Time PCR*

The total RNA of kiwifruit flesh tissue was isolated from three biological replicates of fruit at every 30 days of storage using the RibospinTM Plant Total RNA Purification Kit (GeneAll Biotechnology Co., Ltd., Seoul, Korea) by following the manufacturer's protocol. The extracted RNA was quantified by Nanodrop spectrophotometer (Thermo Scientific™ NanoDrop 2000, Waltham, MA, USA). One microgram of total RNA was used for cDNA synthesis using the TOPscriptTM RT DryMIX (*dT18 plus*) Kit (Enzynomics; Republic of Korea) according to the manufacturer's protocol.

The real-time qPCR was performed with gene-specific primers (Table S2). Each reaction was performed in triplicate. For each sample in 20 μL final volume were contained 1 μL cDNA, 1 μL specific primers, and 10 μL of 2X Real-Time PCR Master Mix (Including SFCgreen® I in the mixture) according to the manufacturer's protocol (BioFACTTM, Yuseong-Gu, Daejeon, Korea).

Three genes of cell wall modification, *A. deliciosa* polygalacturonase C (*AdPGC*), *A. deliciosa* expansin 1 (*AdEXP1*), *A. deliciosa* expansin 2 (*AdEXP2*), and two genes of ethylene biosynthesis, *A. deliciosa* ACC (1-aminocyclopropane-1-carboxylic acid) synthase 2 (*AdACS2*), *A. deliciosa* ACC oxidase 2 (*AdACO2*) were selected based on our previous studies [28,29]. The lignin metabolism-related genes, *A. chinensis* phenylalanine ammonia-lyase (*AcPAL*), *A. chinensis* cinnamyl-alcohol dehydrogenase (*AcCAD*), and *A. chinensis* peroxidase 2 (*AcPOD2*) were selected according to Li et al. [12]. For cell wall modification genes and ethylene biosynthesis genes quantitative RT-PCR was performed using the Rotor-Gene Q detection system (Qiagen, Hilden, Germany) and included initial annealing of 5 min at 95 ◦C, followed by 50 cycles of 15 s at 95 ◦C, 30 s at 60 ◦C, and 45 s at 72 ◦C [28,29], followed by a melting-curve analysis at the end of each run. The cycling procedure for lignin metabolism-related genes included initial annealing of 3 min at 95 ◦C, followed by 44 cycles of 10 s at 95 ◦C, 30 s at 57 ◦C, and 30 s at 72 ◦C [12]. The kiwifruit actin gene, *A. deliciosa* actin 1 (*AdACT1*) was used as the internal control (housekeeping gene) to normalize expression differences in each sample. Quantitative RT-PCR data were analyzed using the 2−ΔΔCT method [30], and changes in gene expression caused by chitosan treatment was compared to untreated fruit (control) of each sampling date (*p* ≤ 0.05).
