Hieff qpcr green master mix

RNA-seq analysis and assays of detoxification-related CYP450 protein activities in M. separata treated for different times with different concentrations of CGA revealed that MsCYP450 genes and CYP450 detoxification proteins were significantly up-regulated. Therefore, we selected seven MsCYP450 genes that were up-regulated considerably in response to different concentrations of CGA for validation by qRT-PCR using the same RNA that was used in RNA-seq. Primer 5 was used to design specific primer pairs (Table S1), and primers were synthesized by Tsingke Biotechnology Co., Ltd. in China. The cDNA synthesis reaction was performed using the HiScript^® II Q RT SuperMix kit with gDNA wiper (Vazyme, China) according to the manufacturer’s protocol using 1 μg of total RNA as a template per reaction. QRT-PCR was performed with Hieff^® qPCR Green Master Mix (Yeasen, China). Finally, data were analyzed using the 2^-ΔΔCT method (Sun et al., 2017 (link)), and EF-1α was used as a control to correct for sample-to-sample variation. Three technical replicates were performed for each replicate, and the data were expressed as mean ± standard error (SE).

Total RNAs were separately collected from samples and then converted to cDNAs for each miRNA by RT-primer (Table S1) using HiScript^® III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The quantitative real time-PCR (qRT-PCR) was performed using the Hieff q-PCR Green Master Mix (Yeasen, Shanghai, China) to examine the expression of miRNAs. The relative levels of miRNAs were normalized to the level of U6 snRNA using the ^ΔΔCT method [78 (link)]. The results were analyzed from three independent samples. The primer sequences for these miRNAs are listed in Table S1.