Quantifying Gene Expression in Kiwifruit

Total RNA was extracted through a Quick RNA isolation Kit (Huayueyang Biotechnology, Beijing, China). RNA quality was evaluated by agarose gel electrophoresis and using the NanoDrop system (Implen, Los Angeles, CA, USA). cDNA was synthesized with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Inc., Dalian, China).
Full-length sequences of genes, including CS, Aco, IDH, SDH and DQS were searched in kiwifruit gene database, and qRT-PCR primers were designed by Primer 5.0 software, according to them [48 (link)]. The qRT-PCR primers were synthesized by Sangon Biotechnology, Shanghai, China (Table 2). The qRT-PCR mixture (20 μL) contained 10 μL TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Inc., Dalian, China), 0.5 μL of the forward and reverse primers for each gene, 1 μL cDNA template, and 8 μL ddH₂O. The LightCycler^® 480 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) was used to conduct the reaction. The conditions for the qRT-PCR amplifications were as follows—95 °C for 5 min, followed by 40 cycles of 5 s at 95 °C 30 s at 60 °C and 20 s at 72 °C. The β-actin in the kiwifruit was considered as the reference gene for normalization [49 (link)]. All analyses were repeated three times using biological replicates. The relative expression levels were calculated using the 2^−ΔΔCT method [50 (link)].