Quantitative Analysis of Rice Gene Expression

Total RNA was isolated from rice leaf samples (100 mg tissue per sample) using Trizol reagent (Invitrogen, Gaithersburg, MD, USA). Concentration of total RNA in each sample was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using one μg total RNA per 20 μL reaction using the PrimeScript™ RT reagent Kit with a gDNA Eraser (Takara, Dalian, China). qPCR was then performed using the SsoFast EvaGreen® Supermix (Bio-Rad) on a Bio-Rad iQ5 qRT-PCR system. The expression levels of OsUBC and OsActin1 were determined and used as internal controls as previously reported (Fang et al. 2015 (link); Lu et al. 2016 (link)). qPCR primers specific for RBSDV P10, OsNOA1, OsNIA2, OsPR1b, OsWRKY45 or OsICS1 are listed in the Supplementary Table 1. The qRT-PCR results were calculated using the 2^-ΔΔCt method reported previously (Livak and Schmittgen 2001 (link)).