Applied biosystems 7500 instrument

The cells were pre-treated with Brevinin-1GHd (1, 2, and 4 μM) for 30 min and were cultured with MDEM medium containing 100 ng/ml LPS for 8 h at 37°C. The cells lacking LPS and Brevinin-1GHd treatment were regarded as the negative control. Cells were lysed with trizol and isolated with chloroform, isopropanol to obtain total RNA. After the first-strand cDNA was synthesized by reverse transcription kit (Takara Company, Dalian, China) in light of the manufacturer’s manual, qRT-PCR was performed using an Applied Biosystems 7,500 instrument (Applied Biosystems, America) for estimation of mRNA expression of IL-1β, IL-6, TNF-α, and iNOS. qRT-PCR procedure and data analysis were performed as previously reported (Zeng et al., 2018 (link)). All experiments were performed with at least three repeats. The primer sequences used for the different tested genes are shown in the Supplementary Table S1.

PCR was performed using a fluorescent probe to detect the MTHFR C677T polymorphism. First, genomic DNA was extracted as described above. A total of 10 μL of the PCR reaction mixture (Taq DNA polymerase, PCR buffer, dNTPs, and MgCl₂), 0.25 μL each of the probe and primers and 5.75 μL of double-distilled water were added to a centrifuge tube. Then, 4 μL of genomic DNA was added. The following primer sequences were used for amplification of the MTHFR gene: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ (forward) and 5′ -AGGACGGTGCGGTGAGAGTG-3′ (reverse). The genotyping procedure was conducted according to the manufacturer's instructions (AUSA Pharmed Co, Ltd., Shenzhen, China). The following PCR protocol was used: one predenaturation step for 2 min at 50°C, followed by 45 cycles of a denaturation step at 95°C for 2 min, a reaction step at 95°C for 15 s, and a polymerization step at 60°C for 1 min. The reactions were performed in an Applied Biosystems 7500 instrument (Applied Biosystems, USA). After PCR amplification, the fluorescence signal at 60°C was used to analyze the MTHFR C677T polymorphism. The X-axis represented the “VIC signal” (C allele), and the Y-axis represented the “FAM signal” (T allele). When interpreting the results, the MTHFR C677T polymorphism was categorized as the CC (allele X), TT (allele Y), or CT (both alleles) genotype.

Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, U.S.A.) according to the manufacturer’s instructions. RNA was reverse transcribed with the mRNA First-Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) for mRNA. Quantitative RT-PCR (qRT-PCR) assay was performed on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, U.S.A.) by using SYBR Green (Invitrogen, Carlsbad, U.S.A.). Relative RNA quantification was calculated via the comparative 2^−ΔΔC_t method. The relative expression of indicated genes was normalized to that of GAPDH in each sample.

Total RNA from PBMCs of DVT patients and healthy controls or the experimental cell lines was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, United States). miRNAs and mRNAs were reverse transcribed using the 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and PrimeScript RT reagent Kit (Toyobo, Osaka, Japan), respectively, according to the manufacturer’s instructions. The relative expression of each RNA was quantified using SYBR Green (Invitrogen, Carlsbad, United States) based qRT-PCR assay on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, United States). U6 and GAPDH were used as internal controls for miRNA and mRNA expressional normalization, and relative RNA quantification was calculated via the comparative 2^−ΔΔCt method. For mRNA and miRNA expressional analysis, the PCR primer sequences used are shown in Supplementary Tables S2, S3. Unless otherwise stated, the amplification reaction for each sample was conducted in triplicate.

Quantitative real‐time PCR (qPCR) was used to measure the relative mRNA expression for the gene, POMC, similar to as described in Rancourt et al. 8. Commercially available TaqMan^® probe‐based gene expression assays were used (Life Technologies, Carlsbad, CA, USA) and were run on an Applied Biosystems 7500 instrument according to the manufacturer's protocol (Applied Biosystems, Waltham, MA, USA). Expression levels were normalized to the housekeeping gene BETA ACTIN. When possible exon‐spanning primer sets were selected, qPCR was performed as duplex qPCR with housekeeping gene. Assays were carried out in triplicate, and relative gene expression was calculated using the 2−ΔCT method corrected for the amplification efficiency calculated from standard curves for all primer sets 8, 15, 32. TaqMan^® gene expression assays: POMC: Gg03352057_m1 and BETA ACTIN: Gg03815934_s1, VIC‐labeled, primer limited.

The RT-PCR primers for simple visual detection of HCV mRNA were modified from a previous study using a pair of specially designed primers for the 5′-untranslated region (5′-UTR) of HCV [18 (link)]. The generated product of the HCV amplicon was 157 bp long (Table 1).
The forward primer was biotinylated at the 5′ end, and the reverse primer was labeled with digoxigenin at the 5′ end. They were suitable for capture by streptavidin-coated magnetic particles and anti-digoxigenin antibody-coated nanoparticles, respectively.
The RT-PCR was performed according to the manufacturer’s instructions using the QIAGEN Taq PCR Master Mix Kit (Qiagen). Briefly, 5 μL of cDNA was amplified in a 25 μL reaction mixture containing 12.5 μL of QIAGEN Taq PCR Master Mix (Qiagen), 1 μL of biotinylated forward primer, 1 μL of digoxigenin-labeled reverse primer, and distilled water. Thermal cycling was performed on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster City, CA, USA) under the following conditions: 94 °C for 5 min; 40 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s; and final extension at 72 °C for 10 min. Agarose gel (2%) electrophoresis was used to detect the HCV PCR amplicons.
The performance of the detection system was evaluated in comparison with a clinical real-time PCR test (Cobas HCV) for the detection and quantification of HCV RNA.