Rice Leaf RNA Extraction and Gene Expression

Total RNA was isolated from rice leaf samples (100 mg tissue per sample) using TRIzol reagent (TIANGEN Biotech, Beijing, China). The concentration of total RNA in each sample was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using 1 µg total RNA per 20 µL reaction using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative RT-PCRs were performed on an ABI PRISM 7500 device using a SYBR Premix ExTaq RT−PCR Kit (Takara). Relative transcript levels were calculated by the 2^-ΔΔCT method as previously described (Rao et al., 2013 (link)), and the rice ubiquitin gene (Os03g0234350) was used as an internal control. The primers for quantitative RT−PCR analysis are listed in Table S1.