Lightcycler 96 software 1

Total RNA from MKE-treated HGF-1 cells with or without LPS-PG and RAW264.7 cells treated with or without RANKL were isolated using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). Complementary DNA (cDNA) was synthesized using an All-in-one 5x cDNA Master mix (CellSafe, Yongin, Republic of Korea) according to the manufacturer’s instructions and reacted at 25 °C for 5 min, 42 °C for 15 min, and 85 °C for 5 s. The synthesized cDNA was then reacted with SYBR Green Master Mix (Elpis Biotech, Daejeon, Republic of Korea) for qPCR, and the primer information is shown in Table 1. The gene expression levels were determined from the 2^−ΔΔCq method and automatically calculated using LightCycler 96 Software 1.1 (Roche Diagnostics, Basel, Switzerland). Relative gene expression levels were normalized and quantified using GAPDH.

The reaction mixture of PTP2-qPCR was formulated on ice according to the description of Hieff^® qPCR SYBR Green Master Mix kit (Yeasen, Shanghai, China). The final concentration of EHP-PTP2-F and EHP-PTP2-R primers was 0.2 μM in the optimized reaction system (Table 2). The other components including 2 × Hieff^® qPCR SYBR Green Master Mix 5 μL, standard plasmid DNA template (1.0 × 10³ copies/μL) 1 μL, and nuclease-free water were added to make a total volume of 10 μL. The amplification reaction was performed in the LightCycler^® 96 (Roche, Indianapolis, IN, USA) and the optimized reaction procedure was 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The data analysis was performed using the LightCycler^® 96 Software 1.1 (Roche).

DNA samples were isolated from G. parasuis clinical isolates using the TIANamp Bacteria DNA Kit (Tiangen, China) according to the manufacturer’s instructions. The HRM primers (P1, P2) were designed to amplify the sequences (approximately 100 bp) surrounding the target site. The annealing temperatures of the HRM primers were optimized to approximately 60°C and purified by HPLC.
A sample of 20 ng of the genomic DNA of a G. parasuis strain was used as a template in a 20 μl PCR reaction using a LightCycler® 480 High Resolution Melting Master kit (Roche), with 1× LightCycler® 480 High Resolution Melting Master Mix, and final concentrations of 200 nM of forward and reverse primers and 3 mM MgCl₂. A total of 108 bp PCR products surrounding the target site were amplified in the LightCycler® 96 instrument (Roche), using the following cycling conditions: pre-incubation 10 min 95°C, 45 cycles 3-step amplification (95°C for 10 s, 57°C for 10 s, 72°C for 20 s) followed by one cycle of high resolution melting [95°C for 60 s, 40°C for 60 s, 65°C for 1 s, and finally using a ramp rate 0.07°C/s and an acquisition mode setting of 15 readings/s to reach the target temperature 95°C (1 s)]. Raw data were analyzed using the LightCycler® 96 software 1.1 (Roche), which sorted the samples into groups based on their normalized melting curves. Each sample included 2–3 technical replicates.

Of the identified miRNA profile, five miRNAs (miRNA-126-3p, miRNA-191-5p, miRNA-223-3p, miRNA-363-3p and miRNA-495-3p) were selected for validation. The validation of miRNAs expression in serum was performed by reverse transcription and real-time quantitative RT-PCR (Qiagen). Shortly, 1 μl of RNA eluate was reverse transcribed in 10 μl reaction using the miRCURY^® LNA^® RT Kit (Qiagen) according to the manufacturer’s instructions. The cDNA was diluted 30-fold, 3 μl of this diluted cDNA was used as a template and assayed in 10 μl PCR reactions according to the protocols of the miRCURY^® LNA^® miRNA PCR Assay (Qiagen). The amplification was performed in a LightCycler^® 96 Real-Time PCR System (Roche). qPCR thermocycling conditions were as follows: 95°C for 120 s, followed by 45 cycles of 95°C for 10 s and 60°C for 1 min. Melt curve analysis was performed between 60 and 95°C at a ramp rate of 0.22°C/s. The amplification curves were analyzed using the LightCycler 96 Software 1.1 (Roche). The expression levels of miRNAs were calculated by using 2^–CT and reciprocal ratios were performed [Ratio miR-A/miR-B = log₂ (2^–CTmiR–A/2^–CTmiR–B)], as previously described (Pérez-Sánchez et al., 2018 (link)).