Quantitative RT-PCR Analysis of Plant Transcripts

Total RNA from maize or Arabidopsis was extracted using RNeasy Plant Mini Kit (Qiagen, Germantown, MD, United States) and treated with rDNaseI (Thermo Fisher, Waltham, MA, United States) per manufacturers’ instructions. First strand cDNA was synthesized from 1 μg of total RNA and oligo (dT)₂₀ primers using Superscript III First Strand cDNA synthesis system (Invitrogen, Carlsbad, CA, United States). For qRT-PCR analysis, SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, United States) was used on an Applied Biosystems Mx3000P thermocycler with 10 ng (for maize) or 5 ng (for Arabidopsis) of cDNA as template in a 10 μL reaction. Primer sequences are described in Supplementary Table 1. Relative expression levels compared to internal reference ZmACTIN (Louis et al., 2015 (link)) or AtEF1α-A (Aboobucker et al., 2017 (link)) were calculated using the 2^–ΔΔCt method (Pfaffl, 2001 (link)).