The analysis of gene expression of the mRNA levels of interest was performed by qRT-PCR in the StepOnePlus thermocycler (Applied Biosystems) with the TaqMan Gene Expression Assays system (Applied Biosystems).

The probes and primers for the C5AR1 (Rn02134203), ICAM 1 (Rn 00564227), iNOS (Rn 00561646), VEGF (Rn 01511602) and for the endogenous control ACTB (Rn 00667869) were acquired from the company list of inventoried assays Applied Biosystems.

Real-time polymerase chain reaction was performed in duplicate for each sample using: 10.0 μL TaqMan Universal Master Mix II 2X, 1 μL TaqMan Gene Expression Assay 20 × and 4 μL of diluted cDNA (dilution 1:5) in a final volume of 20 μL, in 96-well plates covered with optical sealant.

The reaction conditions were as follows: temperature of 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.

To calculate the expression level of each target gene, the GenEx Standard 6.1 software (MultiD Analyzes AB) was used, which uses the 2-delta delta Ct method for relative quantification, where Ct (threshold cycle) is the qRT-PCR, in which the amplification reaches the logarithmic phase, where delta Ct is the difference in expression between the target gene and endogenous control of a given sample and 2 ^ delta delta Ct values corresponds to the difference between the 2 ^ delta delta Ct of the sample and the 2 ^ delta delta Ct of control.

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