Quantitative reverse transcription polymerase chain reaction
(qRT-PCR)
Quantitative reverse transcription (RT)-PCR was performed with RNA fromArabidopsis shoots and roots in two completely independent
biological experiments (three RNA biological replicates each) to
synthesize the complementary DNA strand.. The RT reaction was performed
with random hexamers, using the RETROscript® First Strand Synthesis Kit
(Applied Biosystems-Life technologies, Carlsbad, CA, USA). Quantitative
PCR was carried out with 50 ng single-stranded cDNA in a final volume of
20 μL, containing 10 μL of SYBR-Green Master Mix (Applied
Biosystems-Life Technologies) and 250 nM forward and reverse specific
primers (Life Technologies, Supplementary Table 1), using a Real-Time
7000SDS Termocycler (Applied Biosystems-Life Technologies), with
denaturation at 95 °C for 10 min, 40 cycles of 15 seconds at 95 °C and 1
min annealing and extension at 60 °C. Gene expression was quantified by
using the relative 2−ΔΔCt method (Livak & Schmittgen, 2001), and the
glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH ) as
reference (Montero-Palmero et al., 2013).