Cfx connect real time pcr detection system

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 expression level of BnMORF genes under heat and IAA treatments was examined by RT-qPCR. Brassica napus ZS11 plants were grown on 1/2 LS solid medium under long-day conditions with 100 µmol·m^− 2·s^− 1 light intensity at 22 °C for 14 days. For heat treatment, seedlings were placed at 38 °C for 3 h, followed by recovery at 22 °C for 12 h. For IAA treatment, 14-day-old seedlings were treated with 10 µM IAA for 3 h. Following the treatment, root and leaf samples were collected, respectively. Root and leaf samples collected from untreated plants were served as controls [31 ]. Total RNA was isolated using the RNAprep Pure Plant Kit (DP432, Tiangen, China). The first-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (R312-02, Vazyme, China) with the addition of Oligo (dT)₂₀VN and random hexamer primers. The primers used in RT-qPCR analysis were listed in Additional file 8: Table S7. RT-qPCR was performed using the Bio-rad CFX Connect Real-Time PCR Detection System and the ChamQ Blue Universal SYBR qPCR Master Mix (Q312-02, Vazyme, China). PP2A (Protein phosphatase 2 A subunit A3), which showed suitability across multiple conditions, was used as the reference gene [40 (link)]. The relative expression level of each gene was calculated by the 2^−ΔΔCt method [41 (link)].