RNA extraction, cDNA synthesis, and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR)

Total RNA extraction of PBMC was performed using TRIzol reagent (TRIzol reagent; Life Technologies; Frederick, USA) in accordance with manufacturer’s guidelines. The PMN samples were submitted to RNA extraction through TRIzol and chloroform gradient, after the RNA obtaining the samples were passed through a column of the kit mini prep Direct-zol (ZYMO Research, USA) for purification in accordance to manufacturer’s guidelines. During the PMN extraction, the RNA was treated with DNase I included in the kit (mini prep Direct-zol) for 15 min at room temperature. Total RNA extracted from both cells was separately eluted with 20 µL of RNAse free water. Concentrations and purity of RNA in extracts were evaluated using spectrophotometry (NanoVueTM Plus Spectrophotometer, Ge Healthcare. UK) and absorbance ratios values (260/280) ranged between 1.7 and 2.

Before the reverse transcription, the isolated RNA from PBMC was treated with DNase I (DNaseI, Amplification Grade; Life Technologies) for genomic DNA contamination as per manufactures instructions. We synthesized complementary DNA (cDNA) from 500 ng (PMN) and 1000 ng (PBMC) of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). A master mix (10 µL) containing random primers, reverse transcriptase enzymes and deoxynucleotides were added to 11 µL of samples. Samples were incubated at 25 °C for 10 min and then at 37 °C for 2 h, subjected to reverse transcriptase inactivation at 85 °C for 5 min, and stored at − 20 °C until qPCR analysis. The final RT reaction was diluted 1:80 and this cDNA was used as a template for each qPCR reaction.

Analyses of relative abundance of transcripts were performed using SYBR Green PCR Master Mix (Life Technologies) for the amplification reactions in a Step One Plus thermocycler (Applied Biosystems Real-Time PCR System; Life Technologies). Samples were run in triplicate and the maximum CV accepted among the replicates was 0.1. The optimized primer pairs were designed using primer design platform from national center of biotechnology information (NCBI) (https://www.ncbi.nlm.nih.gov/tools/primer-blast) based on the mRNA sequence of target genes obtained from the RefSeq database, on Genbank (https://www.ncbi.nlm.nih.gov/genbank/) and the specificity of the primer were checked by BLAST (NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi). The qPCR products were submitted to SANGER-DNA sequencing, and identities of target genes were confirmed. Details of primers are provided in Table ​Table3.3. In order to select reference genes, the Normfinder Microsof Excel applet was used⁶⁴. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Actin Beta (ACTB) were the most stable genes in PMN and GAPDH and Ciclofilin (PPIA) were the most stable genes on PBMC, therefore, were selected as reference genes among the others evaluated (Ribosomal Protein L30 [RPL30], Ribosomal Protein L15 [RPL15] and 18 S Ribosomal RNA [18S]).

Bovine specific oligonucleotide forward and reverse primer sequences (5′-3′), primer efficiency in the standard curve and amplicon length of the genes evaluated on qPCR.

Determination of qPCR efficiency and Cq (quantification cycle) values per sample were performed with LinRegPCR software. Quantification was obtained after normalization of the target genes expression values (Cq values) by the geometric mean of the endogenous control expression values⁶⁵. Twenty target genes were evaluated on PBMC cells. For PMN, genes with an increased expression in P or NP groups in PBMC were selected and only those validated in this immune cell type were evaluated (n = 9).