Qrt pcr kit

NPCs and NIH3T3 cells were lysed and total RNA was extracted with RNAeasy kit (QIAGEN). The levels of ADAM10 mRNA were determined with qRT-PCR kit (Takara RR036A and RR820A). GAPDH gene was used as internal control. The primers used were: Adam10:5′-cctgccatttcactctgtcattta-3′ & 5′-gtgcccgggctccttcctctactc-3′; GAPDH: 5′-acagcaactcccactcttccacct-3′ and 5′-ttgctcagtgtccttgctgggg-3′. Hes1, Hes5 and β-actin primers were adopted from a previous report40 (link).

P. capsici susceptible to conventional treatments was isolated and purified from a stem base of the diseased pepper in Henan Province of China. Single-spore isolate was obtained and cultured for 3 days at 25 °C on V8 agar plates in full darkness for subsequent experiments, in which V8 agar medium was composed of 100 mL filtered V8 juice, 900 mL deionized water, 1.5 g CaCO₃ and 20 g agar. V8 medium, which was consisted of 100 mL filtered V8 juice, 0.02 g CaCO₃ and 900 mL deionized water, was prepared for a large number of mycelia of P. capsici.
Technical-grade CA was purchased from Sigma and its assay was ≥95%. It was dissolved in acetone and Tween 80 to 3 mg/mL for subsequent experiments, in which acetone and Tween 80 were used as solvents. The medical solution was stored at 4 °C in the dark. qRT-PCR Kit and fluorescence quantitative PCR kit were purchased from TaKaRa [TaKaRa Biotechnology (Dalian) Co., Ltd., China]. Q5 Hot Start High-Fidelity2 × Master Mix was purchased from New England BioLabs (United States).

We tested four different genes with m6A methylation modification for qRT-PCR analysis, they are related to muscle growth and development (Kee and Hardeman, 2008 (link); Otten et al., 2012 (link); Flix et al., 2013 (link); Siddique et al., 2016 (link)). We validated the methylation-modified differential genes and used a qRT-PCR kit (Takara, Dalian, China) to reverse-transcribe the total RNA extracted from the muscle into cDNA. SYBR Green (Vazyme-Q711, China) was used to perform real-time fluorescent quantitative PCR according to the instructions. The ACTB gene was used as an internal reference gene to standardize the expression level of genes. Three trials were performed on three LYWC and three SIM muscle samples. Primer 5 was used to design four pairs of primers, the primer list is shown in Table 1. All primers span the end of the gene. The relative expression of differentially expressed genes was calculated by the 2^−△△Ct method. The data are expressed as the mean ± standard error (sample number n = 3). The t-test in SPSS statistical software (version 22.0, Chicago, IL, United States) was used to perform the statistical analyses in the two groups, and the difference was significant when p < 0.05.