Quantitative Real-Time PCR for Gene Expression

Real-time RT-PCR was performed using hydrolysis probes and the 2x Taqman Universal PCR Mastermix with an ABI Stepone Plus Cycler (Applied Biosystems, Darmstadt, Germany). Reactions were performed in duplicates in a volume of 10 μl with specific primers and probes (SIGLEC7 Hs00255574_m1 (exon spanning probe; PCR efficiency 95%), PPIA Hs99999904_m1 PCR efficiency 100%, β-Actin Hs99999903_m1 (exon spanning probe; PCR efficiency 94%), Tubulin Hs00362387_m1 (exon spanning probe; PCR efficiency 99%), all Applied Biosystems, Darmstadt, Germany). Primer efficiencies were tested in dilution series of a single exemplary investigated blood sample. For all amplicons, a cDNA component control and a control without reverse transcriptase were performed. Cycling program: 20 s 95°C and 50 Cycles: Denaturation 1 s 95°C followed by 20 s 60°C annealing and elongation. Results are presented as C_T or RQ to a calibrator (ΔC_T) [18 (link)] (StepOne Software Version 2.2.2). Real-Time PCR data were efficiency corrected and normalized either to individual reference or to mean reference gene expression [19 (link)].