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.