Peqgold trifast solution

Human PBMC, γδ T cells, Panc89, and PC-3 cells were harvested from co-culture experiments as described above. Effector or target tumor cells were washed once with cold Dulbecco's PBS, resuspended in peqGOLD TriFast solution (#30-2010, Peqlab) and stored at −80 °C until further use. RNA was extracted and transcribed into cDNA using the cDNA synthesis kit (#8994-A30, AmpTec). For PCR amplification, respective PCR primers for NKG2D receptor and ligands were used at the annealing temperature 60 °C (details in Table 1). qPCR data were analyzed using relative quantitation method by normalizing with the mean Ct value of the housekeeping genes (β2-microglobulin, β-actin, and 18S RNA). The calculated relative expression values of NKG2D receptor and ligand genes were imported and visualized as heatmap based clustering using CIMminer (https://discover.nci.nih.gov/cimminer/home.do). The Euclidian distance method with average linkage rule was used (28 (link), 29 (link)).

RNA isolation was performed with peqGOLD Trifast™ solution (PEQlab). Whole RNA (1-4 μg) was reversely transcribed using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas). Quantitative real-time PCR was performed using a Mastercycler ep realplex™ (Eppendorf). Gene expression was normalized to RS14.

Total RNA was extracted from cells using peqGOLD TriFast solution (peqlab Biotechnologie GmbH, Erlangen, Germany) according to manufacturer’s instructions. A total of 2 μg of RNA were reverse transcribed to cDNA in a 20 μl reaction using the High Capacity RNA-to-cDNA Master Mix kit (Applied Biosystems by Life Technologies, Carlsbad, USA), following manufacturer’s instructions. The resulting cDNA was used as template for the quantitative PCR. Quantitative PCR was performed in triplicate, in a total volume of 20 μl, using the primers listed on supplementary table 4 (Table S4), and Fast SYBR Green PCR Master Mix (Applied Biosystems by Life Technologies, Carlsbad, USA), according to manufacturer’s instructions. The relative amounts of the PCR products were analysed using the comparative RQ method and using PPIA gene as an internal normalization control.