Sybr premix ex taq qpcr kit

Methylation modification analysis is based on the SELECT approach according to a previous study^{42 (link)}. Briefly, total RNA (1500 ng) was mixed with 40 nM up primer, 40 nM down primer and 5 M dNTP in 17 μL CutSmart buffer. SELECT qPCR was performed with the following program: 90 °C for 1 min, 80 °C for 1 min, 70 °C for 1 min, 60 °C for 1 min, 50 °C for 1 min and 40 °C for 6 min. Afterward, the qRT-PCR was performed using SYBR premix Ex Taq qPCR Kit (TaKaRa). qRT-PCR was performed with the following program: 95 °C, 5 min; 95 °C, 10 s then 60 °C, 35 s for 40 cycles; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s. Primers for SELECT qPCR or qRT-PCR are listed in Table S6, respectively. Ct values of samples were normalized to their corresponding Ct values of control. All assays were performed with three independent experiments.

To investigate the effects of H₂O₂ or the inhibitors on proinflammatory cytokine expression, we used 0.5 mM H₂O₂ to treat the cells or cells were pretreated with different inhibitors (Bay11-7082, SB202190, or PD98059) for 1 h, in the presence or absence of 0.5 mM H₂O₂ for 3 h; cells were then collected for mRNA expression analysis. Messenger RNA levels were determined by real-time PCR as described by [16 (link)]. After washing IPEC-1 cells two times with ice-cold PBS, total RNA was extracted using the RNAiso Plus Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) following the manufacturer's instructions. After purification and quantitation, reverse transcription was performed using the PrimeScript® RT Reagent Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) following the manufacturer's instructions. Quantitative analysis of PCR was carried out on a ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies) using a SYBR® Premix Ex Taq™ qPCR Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). The primer pairs used are listed in Table 1. Results were analyzed by the 2^−ΔΔCT method with GAPDH as the housekeeping gene as GAPDH displayed no variation among all groups [16 (link)]. All samples were run in triplicate. Relative mRNA abundance of each target gene was normalized to the control group.

An overnight culture of VRE_fm CAU369 was diluted 1:100 in BHI and incubated at 37 °C, 200 r.p.m. for 4 h. Bacteria were obtained at exponential phase and treated with or without lefamulin and vancomycin (0.5 × MIC, 1 × MIC, 10 × MIC) for 1 h at 37 °C. Total RNA was extracted using the EASYspin Plus kit (Aidlab, cat. RN4301) and quantified by the ratio of absorbance (260 nm/280 nm) using a Nanodrop spectrophotometer (Thermo Scientific, MA, USA). The qRT-PCR was performed using SYBR premix Ex Taq qPCR Kit (TaKaRa, catalog no. RR820A). Cycling conditions consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. All samples were analyzed in triplicate and the gene 16 S rRNA was used as an endogenous control as described in our previous publication^{44 (link)}. The fold changes of gene expression were analyzed by 2^−∆∆CT method. Genes names and primer sequences used in the qRT-PCR analysis are listed in Table S7. GraphPad Prism 8 was used to plot graphs