qRT-PCR
Total RNA was extracted from leaves of 4-week old transgenic and VC
plants, using the trizole method (Sigma-Aldrich, USA). RNA samples were
reverse transcribed into cDNA, using the first strand cDNA synthesis kit
from Thermo Fisher Scientific, according to manufacturer’s instructions.
Relative expression of different genes was studied using qRT-PCR on ABI
Prism 7500 Sequence Detection System (Applied Biosystems, USA), and the
design of the primers was based on sequence details (Table S2 ).
Adenine phosphoribosyl transferase (APT1) was used as a housekeeping
gene. The relative gene expression data were analysed using the
2-ΔΔCt quantitation method (Livak and Schmittgen,
2001).