2.10. Total RNA Isolation and Analysis of Gene Expression Using Quantitative RT-PCR

For this purpose, we selected 12 salt-related genes, APX [46], CAT [46], CHS [47], SOD [46], GmbZIP62 [48], GmCAX1 [21], GmHKT1 [49], GmNHX1 [18], GmNHX2 [50], GmSALT3 [20], GmSOS1 [51], and POD [46] for the molecular analysis. Further, we downloaded the RNA-seq data of 14 soybean tissues from Soybase (https://soybase.org/) for the screening of the above-mentioned genes. These genes are comprised of the antioxidant enzymes-encoding genes and some salinity tolerance genes. Then quantitative real-time PCR (qRT-PCR) was used to measure the expression of above genes in soybean plants treated with chitosan modified biochar (CMB) with salt stress (0, 40, 80 mM NaCl). Using the RNeasy Plant Mini kit (Qiagen), the total RNA was extracted from plant tissues and purity/concentration of RNA were examined spectrophotometrically at 260 and 280 nm. Then, using Qiagen Reverse Transcription kit the first strand of cDNA was synthesized. The qRT-PCR reaction was done as follows; (a) total volume of 20 μL, (b) 3 μL of cDNA (4 ng/μL), (c) 0.2 μL of each primer (10 pm/μL), and (d) 10 μL SYBER Green qPCR master mix (Qiagen). The procedures for PCR were as follows: 95 °C for 5 min; 45 cycles of 95 °C for 15 s and 58 °C for 1 min. The qRT-PCR analysis was repeated independently with three replicates. Gene-specific primers used for gene amplification were listed in Table S1. Actin was served as a housekeeping gene, and the relative expression levels were determined using the 2^−ΔΔCt method.