One step rt qpcr kit

Total RNA was purified, and RT-qPCR was performed using a one-step RT-qPCR kit (Takara, Tokyo, Japan). We followed a previously described method [19 (link)]. The specific primers used can be found in Supplementary Table S1. The β-actin gene was used as an internal control for RT-qPCR.

To examine the qRT-PCR analysis, total RNA was extracted from frozen fruit tissue using RNAiso-mate Tissue Kit (Tiangen, Beijing, China). The purity and quantity of RNA were assessed by Nanodrop 1000 spectrophotometer (thermoscientic, Beijing, China). The RNA was extracted then reverse transcribed into the first-strand cDNA using a one-step RT-qPCR kit (Takara, Shanghai, China). As per the manufacturer instructions, quantitative RT-PCR (qRT-PCR) was performed using an SYBR green Premix Ex TaqTM kit (Takara) on an ABI 7500 real-time PCR detection system. The expression data were normalized with the tubulin gene as an internal control to investigate the gene expression level [68 (link)]. The sets of all primers used for qRT-PCR analysis were designed on Gen script online software (https://www.genscript.com/tools/, accessed on 4 June 2021) are listed in Supplementary Table S2. The experiments were conducted for three biological and technical replicates and relative expression levels for each gene were evaluated via the 2^−△△CT method [57 (link),69 (link)].

In order to examine the qRT-PCR results, RNAiso-mate Tissue Kit was used to isolate total RNA from frozen fruit tissue (Tiangen, Beijing, China). A Nanodrop 1000 spectrophotometer was used to check the RNA’s purity and quantity (ThermoScientific, Beijing, China). A one-step RT-qPCR kit was used to reverse transcribe the RNA into the first-strand cDNA (Takara, Shanghai, China). The ABI 7500 real-time PCR detection system was used to perform quantitative RT-PCR (qRT-PCR) following the manufacturer’s instructions and an SYBR green Premix Ex TaqTM kit (Takara). As an internal control, the tubulin gene was used to normalize the gene expression data (Chun et al., 2020 (link)). The primer premier software was used to design qRT-PCR primers enlisted in Supplementary Table 7. The relative expression levels for each of the genes were measured using the 2^–ΔΔCT method with three biological and technical replicates (Livak and Schmittgen, 2001 (link); Manzoor et al., 2021a (link)).