First-strand cDNA synthesis and Real-time quantitative PCR
The expression of a set of ABA, stress, hormone and root-development
related genes previously selected (Ferrández-Ayela et al. 2016;
Martínez-Andújar et al. 2020b) was analysed in roots by real-time
quantitative PCR (RT-qPCR). First strand cDNA was synthesised with one
µg of purified RNA using the iScript Reverse Transcription Supermix for
RT-qPCR (Bio-Rad, Hercules, CA, USA). The resulting cDNA was diluted by
adding 40 µL of sterile distilled water.
Primers were designed to amplify 79 to 143 bp of the cDNA sequences as
described previously (Ferrández-Ayela et al., 2016). To avoid amplifying
genomic DNA, forward and reverse primers were designed to hybridize
across consecutive exons, except in the case of SlNCED1 gene .
RT-qPCR reactions were prepared with 5 μL of the SsoAdvanced SYBR Green
Supermix (Bio-Rad, USA), 1 μM of specific primer pairs, 0.8 μL of cDNA
and DNase-free water (up to 10 μL of total volume reaction). PCR
amplifications were carried out in 96-well optical reaction plates on a
CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Three
biological and two technical replicates were performed per genotype and
treatment. The thermal cycling program started with a step of 30 s at
95°C, followed by 40 cycles (5 s at 95°C, 10 s at 55°C and 20 s at
72°C), and a melt curve (from 65°C to 95°C, with increments of 1°C every
5 s). Dissociation kinetic analyses and agarose gel loading and
sequencing of the PCR product was used to confirm its specificity.
Primer pair validation and relative quantification of gene expression
levels were performed using the comparative Ct method (Schmittgen &
Livak 2008). Data were represented as the relative gene expression
normalized to the Ct value for the tomato housekeeping geneSlACTIN2 (Solyc04g011500) as previously described
(Ferrández-Ayela et al., 2016). In each gene, mean fold-change values
relative to the expression levels of WT were used for graphic
representation. ΔCt values were analyzed using SPSS 21.0.0 (SPSS Inc.,
USA) by applying the Mann-Whitney U test for determining statistical
differences between samples (P-value ≤ 0.05).