Lightcycler 480 real time pcr detection system

Total RNA was extracted from cells in different treatment groups using a RNAiso Plus reagent (TaKaRa, Dalian, China) and then converted to cDNA using PrimeScript RT Master Mix (TaKaRa) according to the manufacturer's protocols. qPCR was performed on a Light Cycler 480 Real-Time PCR Detection System (Roche, Basel, Kanton Basel-Stadt, Switzerland) using SYBR Green I Master Mix (Roche). Expression levels of the following genes were analyzed: RUNX2, OPN, NOX1, NOX2, SOD1, and CAT. The expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as a reference. The Ct value of the GAPDH was subtracted from the Ct value of the target gene (ΔCt), and the average ΔCt value of the triplicates was recorded. The relative expression levels of each gene were determined using the 2-ΔΔCt method. Primer sequences used in this study are listed in Table 1.

For absolute qPCR, the pMD-18T plasmid (mycoplasmal sequence inserted) was diluted in a 1:10 serial dilution five times and utilized as the standard template The absolute copy number of mycoplasmas in the mycoplasma-containing cell culture supernatant was determined by comparison with the standard curve, and the multiplicity of infection (MOI) was determined accordingly.
For relative qPCR, the intracellular mycoplasma copy numbers were normalized to host genome copy numbers. The host cells were suspended in 0.01 M PBS and washed five times to remove extracellular planktonic mycoplasma. The genomic DNA was then extracted from total cell extracts and purified as previously described (20 (link)).
qPCR was performed using the LightCycler^® 480 Real-Time PCR Detection System (Roche, Basel, Switzerland) and an SYBR Green Real-Time PCR 2× premix kit (Takara). The reaction conditions were as follows: 15 sec at 95ºC followed by 45 cycles of denaturation for 20 sec at 95ºC and annealing and extension for 20 sec at 60ºC. The melting curve of the products was determined and found to be specific. Primers used for the M. pulmonis 16s–23s gene spacer region were as follows: forward, 5′-GGAGCTGGTAATGCC CAAAGT-3′ and reverse, 5′-ACGTTCTCGTAGGGATAC CTTG-3′. Primers for host cell genome (β-hemoglobin) were as follows: forward, 5′-GAAGAGCCAAGGACAGGTAC-3′ and reverse, 5′-CCAACTTCATCCACGTT CAC-3′.

Primers were designed based on CaBBX gene sequences for real-time PCR by using the real-time PCR design tool in Integrated DNA Technologies (IDT, https://sg.idtdna.com/scitools/Applications/RealTimePCR/) (All primers are listed in Table S1). Real-time PCR application was carried out in a LightCycler® 480 Real-Time PCR Detection System (Roche, Hercules, Switzerland) with ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). The constitutive actin gene (Gen Bank accession No. AY572427) was used as an internal control and served as a standard gene for normalizing all mRNA expression levels [50 (link)]. A total of 20 μL reaction system contained 10 μL SYBR Color qPCR Master Mix, 1 μL cDNA samples, 0.4 μL of each primer (10 μM) and 8.2 μL ddH₂O. The PCR thermal cycle conditions were as follows: denaturation at 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 58 °C for 20 s and 72 °C for 20 s. Fluorescence intensities were measured for qRT-PCR at the end of each cycle. A melting curve (61 cycles at 65 °C for 10 s) was performed directly to check for specific amplification. The relative gene expression was calculated by using the 2^-△△Ct method [51 (link)], the experiments were performed triplicate technological repeats. The SPSS statistics software (version 17.0) was used to analyze significant differences [52 ].