Validation of Salt Stress Response Genes

Samples were prepared, and total RNA was extracted using the method indicated above. A total of 16 unigenes that responded to salt stress were chosen for validation. Three independent biological replicates of each sample were used in the analysis. A set of gene-specific primer pairs was designed using Primer3 software [24 ]. Reverse transcription was performed with M-MLV reverse transcriptase (TaKaRa). The relative expression of these genes was determined through qRT-PCR analysis using a SYBR^® Green reaction kit (TaKaRa), with the elongation factor 1-alpha gene as a reference. Then, a Mastercycler ep realplex device (Eppendorf, Hamburg, Germany) was used to run the qPCR assays. The transcription data were calculated using the −ΔΔCt method [25 (link)].