4.7. Expression Analysis of Selected Genes

Quantitative real-time reverse transcription PCR (qRT-PCR) was used to measure the expression of selected genes in rice genotypes under control, saline, and alkaline stress conditions. The leaf samples were collected at two time points (0 and 6 h after stress exposure) and were immediately frozen in liquid nitrogen and stored at −80 °C. Three biological replicates per treatment were used for total RNA extraction using Trizol reagent. RNA quality was assessed using 1.2% agarose gel and RNA quantification was performed using an ND-1000 Spectrophotometer (Thermofisher Scientific, Waltham, MA, USA). RNA samples were treated with PerfeCTa DNase 1 (Quantabio, Beverly, MA, USA) and the resulting high-quality RNA was reverse transcribed into cDNA using the iScipt™ first strand cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA).

The sequences of stress-responsive genes were obtained from the Phytozome database [71], and qRT-PCR primers were designed using Primer Quest (Integrated DNA Technologies, Coralville, IA, USA) (Table 3). EF1α (LOC_Os03g08010) was used as an internal standard for expression normalization. The qRT-PCRs were conducted in three technical replicates using cDNA from the biological replicates and iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) [72]. The expression levels of the genes were determined using the 2^−∆∆CT method [73].

Primers for real time q-PCR study for salt and alkaline stress.