Real-time PCR was performed as we described previously [45 (link)], and the used a locked nucleic acid (LNA) probe-based real-time PCR systems are listed in Table 2 .
Briefly, two μL PCR template was added to 18 μL of the PCR master mix (FastStart Universal Probe Master mix; Roche Diagnostics, Darmsted, Germany) containing 500 nM each of the forward and reverse primers (Generi-Biotech, Hradec Kralove, Czechia) and 100 nM LNA (lock nucleic acid) probe (Universal ProbeLibrary; Roche Diagnostics). Ten minutes’ initial heating at 95 °C was followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s. Samples were incubated and measured in duplicates on an iQ cycler with iQ5 Optical System Software 1.0 (Bio-Rad, Hercules, CA, USA). Cq for genes of interest were normalized to β-actin and cyclophilin A, and the relative mRNA fold change expressions were calculated by GenEx 6.1 software (MultiD Analyses AB, Gothenburg, Sweden) according to the 2−CT method [95 (link)].
Briefly, two μL PCR template was added to 18 μL of the PCR master mix (FastStart Universal Probe Master mix; Roche Diagnostics, Darmsted, Germany) containing 500 nM each of the forward and reverse primers (Generi-Biotech, Hradec Kralove, Czechia) and 100 nM LNA (lock nucleic acid) probe (Universal ProbeLibrary; Roche Diagnostics). Ten minutes’ initial heating at 95 °C was followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s. Samples were incubated and measured in duplicates on an iQ cycler with iQ5 Optical System Software 1.0 (Bio-Rad, Hercules, CA, USA). Cq for genes of interest were normalized to β-actin and cyclophilin A, and the relative mRNA fold change expressions were calculated by GenEx 6.1 software (MultiD Analyses AB, Gothenburg, Sweden) according to the 2−CT method [95 (link)].
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