Quantifying Rice GT8 Gene Expression

Rice salt-responsive (OsGolS1) or cold-responsive GT8 genes (OsGAUT21, OsGATL2, and OsGATL5) from microarray analyses were verified by using qRT-PCR. Primers of these four rice GT8 genes were designed by Primer 5.0 (Table S1). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright^® Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin gene was employed as an internal control [37 (link)]. Three biological replicates (from three independent RNA samples) were used for qRT-PCR. For each biological replicate, three technical replicates were also used. The average threshold cycle (Ct) from three biological replicates was employed to calculate the gene expression fold change by the 2^−ΔΔCT method [37 (link),49 (link)].