The samples of fleshy panicle were macerated using the Tissue Lyser LT apparatus (Qiagen, Germantown, USA), 1.0 mL of Trizol (Invitrogen-Life Technologies, Carlsbad, USA) and stainless-steel beads were added to the microcentrifuge tubes. Fragmentation was carried out for 6 min at 50 Hz.

After removing the beads, 0.2 mL of chloroform (Merck, USA) was added. The samples were centrifuged for 15 min at 12,000 rpm at 4 °C. After centrifugation, the aqueous phase was transferred to a new microcentrifuge tube and 0.5 mL of cold isopropyl alcohol (Merck, USA) was added to precipitate the RNA. The samples were incubated at room temperature for 10 min and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was discarded and the precipitate, containing RNA, was washed with 1.0 mL of 75% ethanol. It was centrifuged for 5 min at 10,000 rpm at 4° C. The flask containing RNA was resuspended in 50 to 100μL of sterile ultrapure water free of DNase/RNase (Invitrogen-Life Technologies, Carlsbad, USA).

The concentration of the extracted RNAs was determined on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA). The degree of purity was evaluated by the ratio 260/280 nm, using only RNAs which ratio was ≥ 1.8. For the analysis of the integrity of the RNAs, electrophoresis in agarose gel was performed to check the 28S and 18S bands. The extracted RNAs were stored at -80 °C until use. The degree of purity of the RNA was confirmed with the average ratio 3 1.9.

For the synthesis of cDNA from the total RNA, the high-capacity RNA-to-cDNA kit (Applied Biosystems) was used in a GeneAmp 2400 thermocycler (Applied Biosystems). In final volume of 20 μL: 1.0 μL of enzyme mix; 10.0 μL of RT buffer; qsp 20 μL of sterile ultrapure water free of DNase/RNase and total RNA (500 ng). For the reaction and stopping the reaction, the tubes were incubated at 37 °C for 60 min and at 95 °C for 5 min, respectively. The cDNA samples were stored at -20 °C until use.

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.

Because of the small sample size, the two groups variables were compared using the Wilcoxon rank sum test (non-normal distribution), considering an alpha p of 0.05 and 80% power. Statistical software STATA version 14 (StataCorp, 2015, Stata Statistical Software: Release 14. College Station, TX: StataCorp LP) was used for calculation.

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