4.4.3. RNA-Seq Data Validation Using qPCR

Total RNA was extracted using a general plant total RNA extraction kit (BioTeke Corporation Co., Ltd. Cat. # RP3301, Wuxi, China). DNA was removed from the RNA sample using the RQ1 RNase-Free DNase kit (Promega Cat. # M6101, Beijing, China) following the manufacturer’s instructions. First-strand cDNA synthesis was carried out using HiScript^® III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd. Cat # R312-01, Nanjing, China) following the manufacturer’s instructions in a 20 µL total reaction volume. The qPCR analysis was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, MA, USA) in a total of 10 µL reaction volume using ChamQ universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Vazyme code: Q711-02, Nanjing, China). The amplification conditions were 50 °C for 20 s, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Three biological and two technical replicates per sample were carried out, and Actin was used as the internal standard. The Ct value was determined using the instrument’s software, and the relative quantification of gene expression was monitored after normalization using Actin. The relative transcription levels were calculated using the ΔΔCt method [69], and leaf was considered as the control tissue for normalized relative expression.