Chamq universal sybr qpcr master mix

Mycelia of ε-PL hyper-yielding strain WG-608 and the original strain M-Z18 were sampled at 96 h during the fed-batch fermentation, then 10 DEGs (sdhA, pls, metH, gltA, aceA, aspB, typB, glk, ppc, ppdk) associated with ε-PL biosynthesis pathway were analyzed by qRT-PCR to ensure the reliability of RNA-sequencing. Total RNA obtained from same individuals was extracted using HiScript III RT SuperMix for qPCR(+gDNA wiper) (Vazyme, China). cDNA was synthesized using AMV First Strand cDNA Synthesis Kit (Sangon Biotech, China) based on the kit instructions, while the specific primers for target genes were designed using Beacon Designer 7 software and listed in Supplementary Table S2. The qRT-PCR experiment was performed as described by Du et al. (2022) (link) using StepOne Real-Time PCR (Applied Biosystems, United States) and SYBRR Premix Ex TaqTM (Takara, Japan) with the following procedures: pre-incubation at 95°C for 30 s; 40 cycles at 95°C for 5 s, 60°C for 30 s, and cooling at 50°C for 30 s. The 20 μL reaction system was composed of 10.0 μL 2 × ChamQ Universal SYBR qPCR Master Mix, 2 μL DNA/cDNA template, 0.4 μL forward and reverse primers (10 μM), and 7.2 μL dH₂O.The housekeeping gene hrdB, encoding RNA polymerase principal sigma factor, was selected as the reference gene for normalization.

Total RNA was extracted utilizing TRIzol reagent (Beyotime, China). In total, 500 ng RNA was reversed transcribed through HiScript II 1st-Strand cDNA Synthesis kits (Takara, Beijing, China). RT-qPCR was carried out via ChamQ Universal SYBR qPCR Master Mix (Takara, Beijing, China) as well as LightCycler 480 instrument. The sequences of primers included the following: ZC3H13: 5′-TCTGATAGCACATCCCGAAGA-3′ (forward) and 5′-CAGCCAGTTACGGCACTGT-3′ (reverse); PKM2: 5′- ATGTCGAAGCCCCATAGTGAA-3′ (forward) and 5′-TGGGTGGTGAATCAATGTCCA-3′ (reverse); and GAPDH: 5′- CTGGGCTACACTGAGCACC-3′ (forward) and 5′-AAGTGGTCGTTGAGGGCAATG-3′ (reverse). Data were quantified with a comparative Ct method (2^−ΔΔCt).

The 31 genes in female and male transcriptomes were validated by qRT-PCR in different tissues and these genes, compared with all candidate genes, were highly expressed based on RPKM. Antennae (480) were collected from female and male adults. RNA was extracted using Trizol reagent and cDNA was synthesized using a PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). We designed gene-specific primers using Primer3. The amplification efficiency was calculated for the genes and two candidate internal reference genes (actin and EF1-a). Based on the results, actin and EF1-a were selected as the reference genes for qRT-PCR. PCR was performed using Mx3000P QPCR (Agilent, United States) and ChamQ universal SYBR qPCR Master Mix (TaKaRa). The PCR program was as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s; and 65–95°C at 0.5°C/5 s. A melting curve was generated. Each reaction had three independent biological replicates, and the relative gene expression level was obtained by the 2^−∆∆Ct method (Guo and Rao, 2008 (link)). The relative gene expression in antennae was analyzed using SPSS v19.0 software. An independent sample t-test was performed to compare the gene expression levels between male and female antennae. Prism v6.0 was used to generate bar graphs.