Evo m mlv rt mix kit with gdna clean for qrt pcr

The RNA-seq data was confirmed by qRT-PCR analysis of fifteen DEGs selected randomly. cDNA was synthesized from 1 μg of total RNA by Evo M-MLV RT Mix Kit with gDNA Clean for qRT-PCR [AG11728, Accurate Biotechnology (Hunan) Co., Ltd], then analyzed by qRT-PCR using Hieff^® qPCR SYBR^® Green Master Mix (High Rox Plus) (Cat No. 11203ES08; Yeasen, Shanghai, China) on StepOnePlus system (Applied Biosystems, United States). BrActin (Bra028615) was used as a quantitative reference (Dheda et al., 2004 (link)). Primer 6.0 software (Premier, Ottawa, Canada) was used to design the corresponding primers, which are listed in Supplementary Table 3. Each 20 μL PCR reaction comprised 10 μL of 2× SYBR Green Master Mix, 0.4 μL of 10 μM primers, 2 μL of cDNA template, and 7.2 μL of ddH₂O. qRT-PCR was as follows: 95°C 5 min, 95°C 10 s, 60°C 30 s, 40 cycles. Each sample had three biological and three technical duplicates. The 2^–ΔΔCt approach was applied to quantify relative gene expression levels (Schmittgen and Livak, 2008 (link)).

Total RNA was extracted using the EASYspin Plus Complex Plant RNA Kit (RA53, Aibosen Biology, Beijing, China), and it was detected by agarose gel electrophoresis. First-strand cDNA was synthesized according to the instructions of the Evo M-MLV RT Mix Kit with gDNA Clean for qRT-PCR (AG11728, Accurate Biology, Hunan, China). Finally, using the SYBR Green Premix Pro Taq HS qRT-PCR Kit (AG11701, Accurate Biology, Hunan, China) [33 (link)], qRT-PCR detection was performed on a CFX Connect^TM Real-Time PCR Detection System (PXF2080, Bio-Rad, CA, USA). Spβ-actin was used as an internal reference gene, and the primer sequences are listed in Table S1. The quantitative results were analyzed using the 2^−ΔΔCT method [39 (link)].