Quantitative RT-PCR for Gene Expression

Total RNA was extracted from the leaves, roots, and Oc cells using RNAiso Plus (Takara, Japan). After combining 3 µg of RNA with 10 ng of oligo (dT), the first-strand cDNA was synthesized using 100 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA), 100 units of RNasin (Promega), and 2.5 mM deoxyribonucleotide triphosphates. The gene expression levels were analyzed by quantitative RT-PCR using SYBR Green I, Prime Q-Matermix (2x) (GENET Bio, Daejeon, Korea) in a Rotor-Gene Q system (Qiagen, Hilden, Germany). Rice Actin1 (OsActin1) was used as an endogenous control to normalize the expression level of the respective genes. All experiments were repeated at least three times, and the relative transcript levels were calculated using the ΔΔCt method (Schmittgen and Livak, 2008 (link); Yoon et al., 2022 (link)). The primers used in this study are listed in Supplementary Table 1.