Total RNA Isolation, High Capacity Reverse Transcription, Pre-amplification of cDNA, and Semi-quantitative Real-Time TaqMan PCR (qPCR)

Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions and concentration and purity were assessed with a NanoDrop 2000C spectrophotometer (ThermoFisher scientific AG Reinach, Switzerland). The elimination of possible genomic DNA contamination was performed using the RQ1 RNA-free DNase Kit (Promega, Dübendorf, Switzerland) following the instructions provided by the manufacturer. Reverse transcription (RT) and pre-amplification of cDNA were performed by applying the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA), using 10 ng total RNA as starting material. Next, the TaqMan PreAmp Master Mix Kit (Applied Biosystems) was applied following the supplier's protocols and as previously described (25). Briefly, all used predesigned commercially available TaqMan systems (obtained from Applied Biosystems) and self-designed primers and 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (TAMRA) labeled probes (ordered from Microsynth AG, Balgach, Switzerland), were pooled. Afterwards, cDNA from each sample was mixed with PreAmp Master Mix and pooled TaqMan assays and samples were amplified in an Eppendorf Mastercycler (Vaudax-Eppendorf AG, Basel, Switzerland). A complete list of the predesigned TaqMan systems and self-designed primers and 6-FAM and TAMRA probes used is presented in Table 1.

List of gene symbols, corresponding gene names, and TaqMan systems used for semi-quantitative real time qPCR.

The expression of the 29 selected target genes was investigated by real-time TaqMan PCR. The construction of self-designed primers and probes was based on published coding sequences (CDS). For genes where only predicted CDS were available (i.e., CD206 and NCR1), products were commercially sequenced (Microsynth) to confirm the specificity of amplicons. Efficiency values of PCR reactions were validated to ensure approximately 100% as previously described (25, 26). The protocols used for sample preparation and semi-quantitative real-time TaqMan PCR were published previously (25–27). TaqMan PCR was run with FastStart Universal Probe Master (ROX, Roche Diagnostics AG, Switzerland) and 5 μl pre-amplified cDNA. Reactions were run in duplicate in an automated ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Autoclaved water and minus-RT controls were used instead of cDNA as negative controls, and relative quantification of gene expression was performed with the comparative Ct method (ΔΔCt), as previously described (26, 27). Values were calibrated to average expression in E– samples and normalized with the expression of reference genes. In preliminary experiments, the expression of three potential reference genes (GAPDH, β-ACTIN and CYCLOPHILIN) was evaluated in all used samples and their stability values were calculated using the online tool RefFinder (28). β-ACTIN and GAPDH were selected as more stable than CYCLOPHILIN and used as reference genes for ΔΔCt evaluation.