Quantitative RT-PCR Analysis of Plant Gene Expression

Total RNA was isolated using an Isolate RNA Minikit (Bioline, Wayne, NJ, USA). The RNA was quantified with a NanoDrop ND-1000 spectrophotometer (ThermoScientific Fisher, Waltham, MA, USA), and 100 ng per RT–qPCR reaction was directly added to an AzuraQuant 1-Step qPCR Mix (Azura Genomics Inc., Raynham, MA, USA), which allows for reverse transcription and real-time PCR amplification in a single tube. Reactions were performed in a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) as described earlier [19 (link)]. B. napus Actin (AF111812; [47 (link)]) mRNA was used as the internal control. For in planta detection of U:PEST expression specific primer pairs were designed that covered the eK and PEST sequences. All experiments were repeated at least three times as biological replicates, if not otherwise stated. The 2(-delta delta C(T)) method was used to calculate relative gene expression [48 (link)]. All primers were ordered from MilliporeSigma, Bedford, MA, USA, and sequences are listed in Table S2.