Aceq qpcr sybr green kit

Real-time PCR analysis was performed according to our previous method [44 (link)]. To measure the expression of hepatic lipogenic gene in the liver samples, total RNA was extracted by RNA-easy Isolation Reagent (Vazyme, Nanjing, China). cDNA was synthesized using HiScript II cDNA Synthesis kit (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was conducted using AceQ qPCR SYBR Green kit (Vazyme, Nanjing, China). The relative quantification was performed using the 2^−ΔΔCt method. The primer sequences are listed in Table S2 of the Supplementary Materials.

A reverse transcriptase quantitative PCR (RT-qPCR) analysis was carried out to study the impact of baicalin-aluminum on the virulence genes expression of C. perfringens. C. perfringens was grown in TGY broth with different concentrations of baicalin-aluminum (ranging from 0 to 50 μg/mL) to an OD₆₀₀ of 0.8 at 37°C under anaerobic conditions. Bacterial RNA was isolated using saturated phenol and purified by TRIzol and chloroform as previously described (Li et al., 2013 (link)). The purified RNA was used to synthesize cDNA using HiScript qRT SuperMix (Vazyme Biotech). The cDNA amplification was manipulated using AceQ qPCR SYBR Green kit (Vazyme Biotech) in an ABI PRISM 7500 Fast Real-time PCR System. The acquired cycle threshold (CT) of each gene was normalized to the CT of the 16S rRNA. The relative amount of mRNA expression levels was calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primers utilized in this study are listed in Table 1.

The mRNA levels of hcp and norV genes in A. hydrophila were measured using qRT-PCR. Bacterial RNA was isolated with an E.Z.N.A. bacterial RNA isolation kit (Omega, Beijing, China). Then cDNA was synthesized in triplicate using HiScript qRT SuperMix (Vazyme Biotech). The cDNA amplification was manipulated using AceQ qPCR SYBR Green kit (Vazyme Biotech) in the Applied Biosystems StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, USA). All procedures above were performed according to the manufacturer’s instructions. The internal housekeeping gene recA was used as the reference, and the acquired cycle threshold (CT) of each gene was normalized. The fold-change of mRNA levels was calculated using the 2^−ΔΔCT method [32 (link)]. The primers used are listed in Additional file 1.