Sybr qpcr supermix

The total RNA was extracted from fresh renal arteries and endothelial cells, using TRIzol reagent (Invitrogen, CA, USA). The RNA concentration and quality were measured using a NanoDrop ND-2000 (Thermo Scientific, Waltham, MA, USA). The First Strand cDNA Synthesis Kit (Novoprotein, Shanghai, China) and the miDETECT A Track miRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China) were applied for reverse transcription. The mRNA levels of eNOS and miR-155-5p were detected via quantitative real-time qPCR, with the application of SYBR^® qPCR Super Mix (Novoprotein, Shanghai, China) and the miDETECT A Track miRNA qRT-PCR Starter Kit. All primers are listed in Table S1 in the Supplementary Materials.

The cDNA templates used for qRT-PCR analysis were generated using the method described above and further amplified with β-actin primers to exclude any possible residual DNA contamination. Primers of Posox9a and Posox9b for qRT-PCR were designed outside the conserved domains to prevent any non-specific amplification. qRT-PCR was performed in a 20 μL system containing 10 ng template cDNA, SYBR qPCR SuperMix (Novoprotein, Shanghai, China), 2.5 μmol/L each of specific forward and reverse primers and RNA-free water. Posox9a, Posox9b and amh amplicons were separated through 1.5% agarose gel electrophoresis, purified using the Zymoclean Gel DNA Recovery Kit, cloned into the pMD-19T Vector. The positive clone was verified by sequencing and used for plasmid construction. A standard curve and amplification efficiency were derived from the serial dilutions of the relative plasmid. Melting curves were generated at the end of each run to assess the specificity of the amplicons and the absence of dimers. A negative control (no template) was always included. The result of absolute quantification for the target genes were calculated by the standard curve method with 18S RNA as the reference gene. All qRT-PCR assays for a particular gene were conducted under identical conditions in triplicate. All primers were listed in Table 2.

THP-1 monocytes were seeded at a density of 1 × 10⁶ cells per mL in 12-well plates. Then E. coli BEVs at a protein concentration of 1μg/mL and S.aureus BEVs at a concentration of 10 μg/mL were added to each well followed by incubation for 6 h. Cells stimulated with PBS served as the negative control.
To measure gene expression of IL-6, IL-8, and IL-1β, total RNA was isolated with an RNA Extraction kit (Tiangen, China) according to the manufacturer’s instructions. 1 μg of isolated RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis SuperMix Kit (Novoprotein, China). Then, 0.5 μl of cDNA was used as the template for each real-time PCR. Real-time PCR was performed using a SYBR qPCR SuperMix (Novoprotein, China) on ABI7500 real-time PCR system (Thermo Fisher Scientific, USA). Primers for target genes are presented in Supplementary Table 1. Gene expression was normalized to GAPDH production in each sample, and the fold induction was determined by using the △△C_T method. Each experiment was performed with triplicate samples and repeated three times.
To determine the production of cytokines, cell culture supernatants were collected. The levels of IL-8 and IL-1β were measured by enzyme-linked immunosorbent assay kits (ELISA, solarbio, China) according to the manufacturer’s instructions. Each experiment was performed with triplicate samples and repeated three times.