The total RNA was isolated from all the samples using the EASYspin RNA plant-kit (Cat#DR103-03) following the instruction manual. DNaseI (RNase-free) was used to eliminate the genomic DNA contamination in the RNA samples. The concentration and purity was checked by Thermo fisher Scientific Nano-Drop One and run on 1% agarose gel. The total RNA (5 g) was taken as a template for a first strand cDNA synthesis using the iScriptTm Reverse Transcription Supermix for RT-qPCR (BIO-RAD, Hercules, CA, USA).
BIO-RAD’s CFX Connect Real-Time PCR Detection System was used to study the relative expression level of the G.barbadense and G. hirsutum NHX genes using the iTAQ UNIVERSAL SYBR GREEN MIX (BIO-RAD) with gene-specific primers. Each gene expression was normalized with the Actin genes [77 (link)]. The thermal cycler conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 1 s, and 72 °C for 30 s, and the melting curve stage was at 95 °C for 10 s, 65 °C for 1 min, and 97 °C for 1–5 s.
BIO-RAD’s CFX Connect Real-Time PCR Detection System was used to study the relative expression level of the G.barbadense and G. hirsutum NHX genes using the iTAQ UNIVERSAL SYBR GREEN MIX (BIO-RAD) with gene-specific primers. Each gene expression was normalized with the Actin genes [77 (link)]. The thermal cycler conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 1 s, and 72 °C for 30 s, and the melting curve stage was at 95 °C for 10 s, 65 °C for 1 min, and 97 °C for 1–5 s.
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