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Real-time RT-PCR

YL Yi Li
BC Boon Huat Cheah
YF Yu-Fu Fang
YK Yun-Hung Kuang
SL Shau-Ching Lin
CL Chung-Ta Liao
SH Shou-Horng Huang
YL Ya-Fen Lin
WC Wen-Po Chuang
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Total RNA samples from another independent experimental batch were used for qRT-PCR. For each sample, 1 μg of total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, USA). Each qPCR consisted of 3 μL PCR-grade water, 5 μL iQ SYBR Green Supermix (Bio-Rad), 0.5 μL forward primer, 0.5 μL reverse primer and 1 μL cDNA template. qPCR was conducted on a CFX Connect Real-Time PCR Detection System (Bio-Rad) following the qPCR kit protocol. Melting curves were generated to confirm primer specificity. Relative expression of a gene of interest [ΔCt; ΔCt = Ct (gene of interest)-Ct (reference gene)] in each sample was measured in four biological replicates and three technical repeats. Primer details for nine genes of interest and a reference gene ubiquitin (Os03g0234200) are shown in Table S8. The reference gene ubiquitin (Os03g0234200) has been used as control for qPCR analysis in previous studies [92–94].

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