qPCR
RNA was extracted from 200 µL sample material (homogenized, if applicable) using the MagNA Pure 96 (or 24) DNA and Viral NA Small Volume Kit or MagNA Pure 24 Total Nucleic Acid Kit (Roche) and eluted in 50 µL elution buffer. 25µL of extracted RNA were subjected to cDNA synthesis in a total reaction volume of 40µL, applying random hexamer primers and 200U M-MLV Reverse Transcriptase (Invitrogen). Synthesised cDNA was diluted 1:1 with H2O to a total volume of 80µL to allow robotic pipetting of 384-well PCR plates.
qPCR was carried out on LC480II real-time PCR thermal cyclers (Roche) in a total reaction volume of 15 µL. The reaction contained 1x PCR buffer, 4mmol/L MgCl2, 1mmol/L dNTP (ThermoFisher) with dUTP (GE Healthcare), 600ng BSA (ThermoFisher), 0.3-1U Platinum Taq Polymerase (ThermoFisher), variable concentrations of primers and probes (available on request), and 5µL of the prediluted cDNA. After 5 minutes at 95°C for Taq DNA polymerase activation, a total of 45 cycles consisting of denaturation at 95°C for 15 seconds and annealing at 60°C for 30 seconds were performed. After the run, data were analysed using the LightCycler software version 1.5.1.62.