Splenic NK Cell mRNA Isolation and Quantification

Splenic NK cells were directly isolated by magnetic activated cell sorting (MACS) using an NK cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). Thereafter, mRNA was isolated by using Dynabeads oligo (dT) (Invitrogen, Thermo Fisher Scientific Inc.) following manufacturer´s instructions. Synthesis of cDNA was performed by reverse transcriptase reaction according to the supplier's instruction as already described (Thermo Fisher Scientific Inc.) as already described^{72 (link)}. The mRNA concentrations of genes were measured by real-time polymerase chain reactions (qTower, Analytik Jena AG, Jena, Germany) using SYBR Green Fluorescein Mix (BioRad, München, Germany) and the specific primers (KiCq- Start Primers, Sigma Aldrich, Supplementary Table S3). For normalization of target gene values, the housekeeping gene peptidylprolyl isomerase A (Ppia) was used. The relative mRNA concentration was calculated using the ΔΔCt method and individual amplification efficiency for each primer, determined by a standard curve with different primer dilutions^{106 (link)}. The mRNA concentrations of genes were measured by realtime detection reverse transcriptase-PCR (iQ5, BioRad) using SYBR Green MIX (BioRad). For determination of mRNA concentration, a threshold cycle (Ct) was obtained from each amplification curve using the software qPCRsoft 3.4 (Analytik Jena AG).