Quantitative Real-Time PCR for Gene Expression

Quantitative real-time PCR was based on the method of Muhammad Ahsan Altaf [77 (link)], and some modifications were performed. According to the manufacturer’s instructions, total RNA was extracted from different treated tomato roots using an RNA simple total RNA extraction kit (TIANGEN, Beijing, China). With the help of agarose gel electrophoresis and a K5800 micro spectrophotometer (KAIAO, Beijing, China), the purity of the extracted RNA was detected. The RNA was then reverse transcribed using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme, Nanjing, China) for complementary DNA (cDNA) synthesis. For the qRT-PCR analysis, cDNA was used as a template, and the SYBR^®GREEN Premix Pro Taq HS qPCR Kit (ROX Plus) (Accurate Biotechnology, Changsha, China) was used in the QuantStudio5 P-qPCR system (Applied Biosystems, Waltham, MA, USA), using 96-well plates for the qRT-PCR.
The detailed information on the primers used in this study is provided in Table 2, and actin is used as the reference gene. The relative expression level of the gene was calculated using the 2^−ΔΔCt method by referring to the Livak [78 (link)] and Schmittgen [79 (link)] method.