Chamq sybr color qpcr master kit

The total RNA of the pear flesh was extracted using RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer instructions. The HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used to synthesise single-stranded cDNA. The Lightcycler^® 480 II System (Roche, Basel, Switzerland) and the ChamQ SYBR Color qPCR Master Kit (Vazyme Biotech Co., Ltd., Nanjing, China) were then used to detect relative gene expression levels. The reaction protocol was as follows: 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. ACTIN was used as an internal reference. All RT-qPCR experiments were performed in triplicate using three biological replicates and three technical replications. Statistical significance was determined using Student’s t-test by IBM SPSS Statistics v21.0. The used primers are listed in Table S1.

Total RNAs were extracted from various tomato tissues using the TRIzol^® 117 reagent (Invitrogen). A reverse transcription kit (Vazyme, Nanjing, China) was utilized to generate single-stranded cDNA using 2 μg of total RNA. Gene expression assays by qPCR analysis were performed using the ChamQ SYBR Color qPCR Master Kit (Vazyme, Nanjing, China). All data had three biological replicates and were analyzed. The internal control was the expression of the Actin gene (Solyc11g005330). Supplementary Data Table S1 lists the primer sequences used in real-time PCR.

Total RNA was extracted from pear using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Beijing, China). The cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Nanjing, China). The Lightcycler^® 480 II System (Roche, Basel, Switzerland) and the ChamQ SYBR Color qPCR Master Kit (Vazyme Biotech Co.) were used to estimate relative gene expression levels under the different treatments. The reaction system (20 μL total volume) consisted of 2 μL template cDNA, 1 μL each forward and reverse primer, 10 μL Supermix, and 6 μL RNA-free water. The reaction protocol was as follows: 95°C for 5 min, then 45 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The Actin gene was used as an internal control. The relative expression level for each gene was calculated with the 2^−ΔΔCt method. Each sample analysis was repeated three times. The primers used are listed in Supplementary Table S1.