Quantitative Real-time PCR Analysis

Total RNA from at least three silenced plants of each gene was obtained by extraction with RIBOzol Reagent. Remnant genomic DNA was removed by DNase I treatment. First-strand cDNA was synthesized from 0.5 µg of total RNA using RevertAid H Minus Reverse Transcriptase and oligo(dT) (Thermo Fisher Scientific, Carlsbad, CA, USA). Real-time qPCR was carried out using QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) and PyroTaq EvaGreen qPCR Supermix (Solis BioDyne, Tartu, Estonia), specific oligonucleotides, and recommended qPCR cycles as follows: initial denaturation for 12 min at 95°C, followed by 50 cycles of 15 s at 95°C and 60 s at 60°C. Specific oligonucleotides were designed using Primer3web version 4.1.0 (https://bioinfo.ut.ee/primer3). Oligonucleotide efficiencies were tested by qRT-PCR using tenfold serial dilutions of the corresponding cDNA. Each biological replicate was run in triplicate. Elongation factor 1-α (EF1α, TC19582), F-BOX family protein (F-BOX, Niben.v0.3.Ctg24993647) and protein phosphatase 2A (PP2A, TC21939) genes were used as endogenous controls (Liu et al., 2012 (link)).