2.4 Total RNA extraction and RT-qPCR
Total RNA from leaves was extracted using Trizol (Invitrogen, USA) in
accordance with the manufacturer’s instructions. First-strand cDNA was
synthesized from total RNA (1 μg) using the HiScript II Q RT for qPCR
(+gDNA viper) kit (Vazyme, China). RT-qPCR was performed on a
QuantStudioTM 6 Flex Real-Time PCR System (Applied Biosystems,
Singapore) using a CHamQ SYBR RT-qPCR Master Mix kit (Vazyme, China),
following the supplier’s protocol. Expression levels were normalized
against expression of the housekeeping gene OsUBQ5 and analyzed
by the comparative Ct method (2-∆∆Ct method) (Livak &
Schmittgen, 2001). At least three biological replicate samples were
used. Differences were considered significant at P <
0.05. The primers used in this study are listed in Table S1.