Quantification of cytokine mRNA by real-time quantitative PCR (qPCR)

Primers used for quantitative real-time PCR for equine IL-2, IL-4, IL-10, IL-18, IFN-γ, IL-6, TNF-α, IL-12p35, and housekeeping gene ß-actin are shown in Supplementary Table-1. Primer sequences against TNF-α, IL-6 [18], and IL-12 [19] genes were taken from the previous studies. Five-fold serial dilutions of known copy number recombinant cytokines clones were used for standard curve preparation as previously described method [20]. The clone of respective cytokine gene in pGEM-T Easy vector was purified with nucleospin plasmid mini kit (Nucleospin, Germany) and nucleic acid concentration was determined by Nanodrop spectrophotometer (Eppendorf, Biophotometer Plus Spectrophotometer, Germany). The number of copies/µl was calculated according to the following formula:

Primer sets for SYBR Green qRT-PCR, target cytokine, and standard curve data.

F=Forward, R=Reverse, E=Reaction efficiency, RSq=Linear correlation, S=Limit of sensitivity, bp=Base pair, CV=Coefficient of variance. qRT-PCR=Quantitative reverse transcription polymerase chain reaction

Where,

C = Concentration of plasmid DNA in g/µl and M.Wt = Molecular weight of cytokine plasmid (base pair 660× g).

PCR reaction consisted of 5 µl of 2× SYBR Green master mix (Clontech, USA), 1 µl of each primer (2 μM), and 1 µl of plasmid DNA in a final volume of 10 µl. A non-template control (RNase-free water) was included for every PCR run. Thermal program was set at 95°C for 3 min for initial denaturation and 40 cycles of 95°C for 5 s and 60°C for 30 s and ran on CFX96^™ real-time system (Bio-Rad, Singapore). A melting curve analysis was performed after the amplification phase to determine any non-specific amplification or primer-dimer formation. Repeatability and reproducibility of the qPCR assays were determined by intra-assay and interassay variation, respectively [18].

PBMCs were harvested at 3 h and 24 h post-stimulation and RNA was isolated using Tri-reagent (Sigma, USA) according to the manufacturer’s instructions. The purity and quantity of RNA were assessed using a NanoDrop spectrophotometer (Eppendorf, Biophotometer Plus Spectrophotometer, Germany). Subsequently, 1 μg of RNA was reverse transcribed using SmartScribe^™ reverse transcriptase cDNA kit (Clontech, USA). Following synthesis, cDNA was stored at −20°C. Real-time quantitative reverse transcription-PCR was performed in 10 µl volume including 5 µl of a 2× SYBR Green master mix (Clontech, USA), 1 µl of each primer (2 μM), and 1 µl of cDNA and nuclease-free water. A non-template control (RNase-free water) and no RT control were included for every PCR run. All reactions were performed in duplicates, and same thermal conditions were followed as mentioned above. The specificity and size of real-time PCR products for each cytokine were confirmed by 2% agarose gel electrophoresis and sequencing of PCR products. Copy number of cytokines in control and stimulated PBMCs was calculated by extrapolating the C_t value to the standard curves.