Eosinophil RNA Analysis with qPCR

RNA from eosinophils was purified using an RNA isolation kit (Macherey–Nagel, Düren, Germany). RNA concentrations were normalized, cDNA was synthesized using the QuantiTect Reverse Transcription System (Qiagen, Venlo, Netherlands) in a Biometra TAdvanced Thermocycler (Analytic Jena, Jena, Germany). cDNA was analyzed with GoTaq qPCR Mastermix (Promega, Fitchburg, USA) using the in Supplementary Table 3 listed primers, Morrbid and Bim primers were analogous to Kotzin et al. (2016 (link)). Reactions were performed and analyzed using a LightCycler 480 Instrument II (Roche, Rotkreuz, Switzerland). The 2^−ΔΔCt method (Livak and Schmittgen 2001 (link)) was used for the relative quantification of the measured gene expression levels. To ensure comparability among the analysis, the housekeeping gene HPRT was included as internal control and the cell lines HL-60/KG-1 alpha were included as calibrator. Serum IL-5 was measured at baseline using a human IL-5 ELISA Kit (Biorbyt, Cambridge, UK). Reactions were performed and analyzed in a GloMax Discover Microplate Reader (Promega, Fitchburg, USA).