Hiscript 2 q rt supermix for quantitative pcr

mRNA was extracted from the cells using RNAfast2000 (Feijie, Shanghai, China) according to the manufacturer’s guidelines, and 100 ng of RNA was used for qRT-PCR experiments. Subsequently, HiScript II QRT SuperMix for quantitative PCR (Vazyme, Nanjing, China) was used for quantitative real-time PCR (qRT-PCR) analysis. The primer sequences used to quantify cDNA through qRT-PCR experiments were documented in Table 1. The relative quantities were determined using the 2^−ΔΔCT method and normalized to β-actin.

The total RNA of human spermatozoa was extracted using the Allprep DNA/RNA/Protein Mini Kit (QIAGEN), and approximately 1 µg of obtained RNA was converted into cDNAs using HiScript II Q RT SuperMix for quantitative PCR (Vazyme). The obtained cDNAs were individually diluted 5-fold to be used as templates for the subsequent real-time fluorescence quantitative PCR, with AceQ qPCR SYBR Green Master Mix (Vazyme) on a CFX Connect Real-Time PCR Detection System. GAPDH was used as an internal control, and the primers for real-time quantitative PCR are listed in Table S2. The expression of mRNA was quantified according to the 2^−△△Ct method.

The mRNA sequences of the wheat GS1 and GS2 genes were obtained from the National Center for Biotechnology Information database (NCBI, GenBank accession numbers: HQ840647 and JF894116) and gene-specific primers were designed using Primer Premier 5.0 software (Table S1). RT-PCR was performed using individual cDNAs obtained from each of the three replicates from the pot experiments, to generate GS1 and GS2 expression profiles. RT-PCR reactions were performed using HiScript II Q RT SuperMix for quantitative PCR (Vazyme Biotech) in a Bio-Rad iQ5 RT-PCR thermal cycler (Bio-Rad, Hercules, CA, USA). The reverse transcription efficiencies of the GS1, GS2, and GAPDH genes were almost the same when the Ct values at different dilutions of cDNA were analysed^{70 (link)}. The reaction was performed using 1 μL of cDNA, 15 pmol of gene-specific primer, and 25 μL of 2 × Q RT SuperMix for a qPCR in a 50-μL reaction volume. The following amplification program was used: 95 °C for 7 min, 40 cycles at 95 °C for 10 s, primer annealing at 58 °C for 30 s, 72 °C for 15 s, 58 °C for 10 s, and 95 °C for 15 s. All samples were amplified in triplicate, and the mean and standard error values were calculated. A completely randomised design was used to analyse the RT-PCR data.