Total RNA isolation and qRT-PCR analysis
Total RNA was isolated using TRIzol reagent (No. 15596026, Invitrogen).
cDNA was synthesized using the PrimeScriptTM RT
reagent Kit with gDNA Eraser (No. RR047A, TaKaRa), according to the
manufacturer’s instructions. qRT-PCR, using SYBR Green PCR mix (No.
RR820A, TaKaRa), was performed using a real-time PCR detection system
(Bio-Rad CFX96) according to the manufacturer’s instructions. The rice
gene ubiquitin was used as an internal reference for all analyses.
Reaction mixtures include a 50–500 ng template, 0.2 μM forward primer,
0.2 μM reverse primer, 10 μL of 2 × Perfect Start Green qPCR SuperMix,
and nuclease-free water to a final volume of 20 μL. The reaction
conditions were as follows: 95 °C for 30 s, then 40 cycles of 95 °C for
5 s, and 60 °C for 30 s. The primers used for the qRT-PCR are listed in
Table S2.