2.7 Validation of DE genes by quantitative reverse transcription
(qRT)-PCR
For qRT-PCR analysis, drought treatment, plant age and sampled leaves
were the same as in the microarray experiments. Total RNA was extracted
from leaf tissue using the TriPure reagent (Sigma–Aldrich), according
to the manufacturer’s instructions, and reverse-transcribed with the
M-MLV enzyme (Invitrogen) as indicated before. The qRT-PCR reactions
were carried out in a Master cycler Rep realplex4thermocycler (Eppendorf) using Platinum Taq DNA polymerase (Invitrogen)
and SYBR Green I (Roche) to monitor the synthesis of double-stranded DNA
under the following conditions: 95°C for 2 min and then 40 cycles of
95°C for 15 s, 55°C for 30 s, and 72°C for 40 s. The relative abundance
of transcripts was estimated with the ΔΔCt method (Schmittgen & Livak
2008), and normalized to the gene encoding elongation factor‑1α (Genbank
accession number AB061263.1 ). Primers used in this study are
listed in Supplementary Table S1. Each qRT-PCR reaction set included
5–6 biological and 2 technical replicate samples, and water used as a
negative no-template control instead of cDNA.