Genomic DNA and RNA preparations, cDNA synthesis, and
quantitative real-time PCR (qPCR)
Genomic DNA was extracted for genotyping as previously reported (Edwards
et al., 1991). Total RNA was prepared with TRIZOL reagent (Invitrogen)
and involved with TURBO DNA-free Kit (Applied Biosystems). cDNA
synthesis was performed with the Ready-To-Go Kit (GE Healthcare Life
Sciences). PCR primers were designed by Primer3 (http://primer3.ut.ee).
qPCR reactions with iQ SYBR Green Supermix (Bio-Rad) were analyzed by
MyiQ thermocycler (Bio-Rad). Data were analyzed with the iQ5 optical
system (Bio-Rad), and normalized to the internal control AtPP2AA3(PP2A ; At1g13320) (Czechowski, Stitt, Altmann, Udvardi, &
Scheible, 2005).