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.