2.3 RNA extraction and qRT-PCR
Total
RNA was extracted using a Foregene Plant RNA Isolation Kit (Foregenes
Co. Ltd., Chengdu, China). cDNA synthesis was performed using a Thermo
Scientific Kit (Thermo Scientific, Carlsbad, CA, USA). qRT-PCR was then
conducted with an SYBR® Premix Ex Taq™ II Kit (Takara,
Kyoto, Japan) and a Roche LightCycler® 96 Real-Time
PCR System (Roche, Indianapolis, IN, USA). Three biological replicates
were used for each experiment. The malate dehydrogenase(MdMDH ) gene was used to standardize gene expression (Table S1).
Relative gene expression was determined using the
2−ΔΔCT method (Livak and Schmittgen, 2001).