Powertrack green master mix

Using the PrimeScript™ RT reagent kit (CAS No.: RR037B, Takara), reverse transcription was performed to transform RNAs into cDNA with high quality and at high quantity. Subsequently, DNA Eraser (Takara) was used to eliminate the genomic DNA in RNA, and then β-actin primers were used as a house gene to evaluate cDNA quality.
The specific DEG primers were designed by Primer Premier 5.0 (Premier Biosoft International, USA; Supplementary Table S1). The cDNA product was subjected to qPCR on the QuantStudio™ 3 Real-Time PCR System (CAS No.: A28136, ABI, United States). Each reaction system included 1 μL cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 5 μL fluorescent dye (PowerTrack Green Master Mix, CAS No.: A46112, ABI, United States), and 3 μL ddH₂O. The reaction was performed under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 amplification cycles (95°C for 15 s and 60°C for 45 s). After amplification, the specificity of PCR was detected using the melting curve, and relative expression was calculated by 2^−ΔΔCt method (18 (link)). Finally, t-test was performed to test for significant differences between genes in the CL and GL groups in SPSS v17.0 (SPSS, Chicago, IL, United States).

The primers of real-time quantitative PCR were designed using Primer Premier 5 (Premier Biosoft, USA). The specific sequences are listed in Table 1. The QuantStudio 3 Real-Time PCR System (ABI, USA) was used for quantitative PCR. The reagents used for real-time quantitative PCR were PowerTrack SYBR Green Master Mix (ABI), and the reaction volume was 10 μL, including 1 μL cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 5 μL PowerTrack Green Master Mix, and 3 μL ddH₂O. The prepared premix was added to a 96-well plate, which was placed on an instrument for amplification. Each reaction was repeated three times to ensure accuracy of the results. The PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s, and 60°C for 45 s. The fluorescence was detected after the extension. A dissolution curve was used after the PCR reaction to detect the specificity of the PCR amplification. β-actin was used as the internal reference gene, and the relative quantitative values of the samples were calculated using the 2^-ΔΔCT method (23 (link)).