One step qrt pcr sybr green kit

The viral RNA in supernatants collected at different time points was extracted using a QIAamp viral RNA minikit (Qiagen, Valencia, CA, USA) and quantified using a One-Step qRT-PCR SYBR green kit (Vazyme, China) with standard curves. For cytokine quantification, cDNA was synthesized using a HiScript II 1st Strand cDNA synthesis kit, and qRT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme).

Total RNA was extracted from a logarithmic-phase bacterial culture using an E.Z.N.A. Bacterial RNA Kit (Omega, GA, USA). cDNA synthesis was conducted using HiScript II QRT Supermix (Vazyme, Nanjing, China). The mRNA transcription levels were examined using a One Step qRT-PCR SYBR Green Kit (Vazyme, Nanjing, China) in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, MA, USA). The fold-change in mRNA expression value was calculated using the 2^−ΔΔCT method and normalized against the expression level of the housekeeping gene recA. The primers used are listed in Table S2. The assay was performed in three independent biological experiments.

One microgram of RNA was reverse transcribed into cDNA using Hiscript II QRT Supermix (Vazyme, Nanjing, China). The mRNA levels of target genes were quantified by qRT-PCR using a One Step qRT-PCR SYBR green kit (Vazyme, Nanjing, China). The primers used for the qRT-PCR assay are listed in Table S3 in the supplemental material. For the transcription of the csbD gene, each group comprises three biological replicates. For the validation of transcriptome results, each group comprises three technical replicates. Data were normalized to that of a reference housekeeping gene (recA) and analyzed by the comparative threshold (ΔΔC_T) or ΔC_T method (54 (link)).

Total RNA was isolated using the RNeasy kit (QIAGEN, United States). cDNA was synthesized using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo Scientific, United States). Stem-loop RT primers (RiboBio, China) were used in reverse transcription for miR-454-3p. qPCR was performed using the One-Step qRT-PCR SYBR® Green Kit (Vazyme Biotech, China). The sequences of primers targeting 4.1N/EPB41L1 and miR-454-3p were used as described earlier (Wang et al., 2010 (link)) and designed by Vazyme Biotech (Nanjing, China). U6 small nuclear RNA was used as an internal control for miR-454-3p analysis.

To validate the transcriptomics results, we used qRT-PCR to measure the transcription levels of randomly selected genes. The primer pairs used are shown in Supplementary Table 2. Total RNA was extracted from stationary-phase bacteria using an E.Z.N.A. bacterial RNA isolation kit (Omega, Beijing, China). cDNA synthesis was performed using HiScript II QRT Supermix (Vazyme, Nanjing, China). The mRNA transcription levels of the 20 genes were examined individually using a One Step qRT-PCR SYBR Green kit (Vazyme Biotech) in an ABI PRISM 7300 Fast Real-time PCR machine. The housekeeping gene recA was chosen as an internal control for qRT-PCR, and the 2^−∆∆Ct method was used to calculate the fold-change of mRNA expression levels.

The intestinal enzyme activity of largemouth bass was determined by a kit. The malondialdehyde (MDA), total protein (TP), glutathione (GSH), catalase (CAT), total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) were all from NanJing JianCheng Bioengineering Institute (Nanjing, China).
Plasma biochemistry was analysed by an Mindray BS-400 automatic analyser (Shenzhen, China). Alkaline phosphatase (ALP), aminotransferase (ALT), high-density lipoprotein (HDL-C), albumin (ALB), low-density lipoprotein (LDL-C) and alanine aspartate aminotransferase (AST) kits were all from Shanghai Zhicheng Biological Technology Co., Ltd. (Shanghai, China).
The relative expression of intestinal genes was detected by fluorescence quantitative PCR (qPCR). A One-Step qRT–PCR SYBR Green Kit (Nanjing Vazyme BioTech Co., Ltd., Nanjing, China) was used for qPCR on a 7500 fluorescence quantitative PCR machine. Table 2 shows the specific primers used in this experiment. Beta-actin (β-actin) was selected as a nonregulatory internal reference gene. The relative expressions were analysed by a relative standard curve method.