Total rna rapid extraction kit

Total RNA extraction was conducted using Total RNA Rapid Extraction Kit (Zomanbio, Beijing, China), and RNase‐free DNase I (NEB) was used to eliminate any genomic DNA contamination. First‐strand cDNA synthesis was performed using PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). qRT‐PCR was conducted using SYBR® Premix Ex Taq™ II (Takara Bio, Inc.), with the following amplification program: one cycle of 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. A previously reported translation elongation factor gene PpTEF2 was selected as the internal control (Tong et al., 2009). Relative gene expression levels were calculated using the formula 2^−∆∆Ct. Three biological replicates were conducted for each sample. Sequences of the primers used for qRT‐PCR are listed in Table S8.

Approximately 100-mg fruit samples were ground into powder in liquid nitrogen and then subjected to total RNA extraction using the Total RNA Rapid Extraction Kit (Zomanbio, Beijing 100085, China) according to the manufacturer’s instructions. RNA extracts were treated with DNase I and then converted to cDNA using the PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara, Dalian 116000, China). qRT-PCR was conducted using TB GREEN (Takara Bio, Inc.), following the manufacturer’s instructions on an ABI StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The amplification program was as follows: One cycle of 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. Translation elongation factor 2 (TEF2) was used as a constitutive control according to the previous report [64 (link)]. All analyses were repeated three times using biological replicates. Primer sequences used for qRT-PCR are listed in Table S1.

Total RNA was extracted using Total RNA Rapid Extraction Kit (Zomanbio, Beijing, China). Removal of genomic DNA contamination and first strand cDNA synthesis were conducted using PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative RT-PCR was carried out in a total reaction volume of 20 μL containing 100 ng of template cDNA, 0.4 μM of each primer, 1 × ROX reference dye, and 10 μL of 2 × SYBR premix Ex Taq II (TaKaRa). The qRT-PCR program was as follows: one cycle of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. A translation elongation factor gene PpTEF2 was selected as an internal control according to the previous report (Tong et al., 2009 (link)). All analyses were repeated three times using three biological replicates. The primers used for qRT-PCR are listed in Supplementary Table S2.