2.5.3 Validation of RNA-Seq data
RNA was extracted using the plant RNA kit (OMEGA) with three
replications. cDNA was obtained using PrimeScript™ RT Reagent Kit
following the manufacturer’s protocol (TaKaRa, Japan) and then utilized
for RT‐qPCR reactions performed in Bio‐Rad CFX96 (Bio‐Rad, USA) using
SYBR® Green Realtime PCR Master Mix (TaKaRa, Japan). Gene‐specific
primers utilized in this study are listed in Table S1. Relative gene
expression levels were normalized utilizing SlUbi3 as a reference
gene and data analyzed using 2−ΔΔCt method
(Livak and Schmittgen, 2001).