RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis

Total RNA was extracted from non-stressed and stressed root tissues of two alfalfa varieties with three biological replicates (each containing 100 mg alfalfa roots) using the RNA simple Total RNA Kit (TIANGEN BIOTECH, Co., Ltd, Beijing, China) based on the kit instructions. cDNA was reverse transcribed from 1 μg of total RNA using a First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) (Niu et al., 2017). Specific RT-qPCR primers were designed using Primer 3⁴ (Table ​Table11). RT-qPCR was conducted with an iQ^TM 5 real-time PCR machine (Bio-Rad, United States) using the SYBR^® Premix Dimer Eraser^TM (TaKaRa, Dalian, China). Each 20 μL reaction mixture contained 2 μL template cDNA, 10 μL of 2× SYBR^® Premix Dimer Eraser, 0.8 μL of each primer (10 μM), 6.0 μL dd H₂O, and 0.4 μL of ROX reference Dye. The amplification reaction conditions were as follows: 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 60°C for 30 s (Niu et al., 2017). The levels of relative gene expression were calculated by the ddCt algorithm using the alfalfa MtGAPDH gene as the housekeeping gene (Zhang et al., 2003). Three biological replicates were performed for each sample.

Sequence of primers used in RT-qPCR.