Iq5 optical system software 1

2 μL of the PCR template was added to 18 μL of the FastStart Universal Probe Master (Roche Diagnostics) containing 100 nM LNA (lock nucleic acid) probe (Universal ProbeLibrary; Roche Diagnostics) and 500 nM each of the forward and reverse primers (Generi-Biotech, Hradec Kralove, Czech Republic)—Table 1.
Ten minutes’ initial heating at 95 °C was followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s 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 the interest was normalized to Cq for β-actin and cyclophilin A reference genes and the relative mRNA fold change expressions of the genes of interest were calculated by 2^−ΔCT method [113 (link)] by GenEx 6.1 software (MultiD Analyses AB, Gothenburg, Sweden).

First, 2 μL of the PCR template was added to 18 μL of the FastStart Universal Probe Master (Roche Diagnostics) containing 100 nM LNA (locked nucleic acid) probe (Universal ProbeLibrary; Roche Diagnostics) and 500 nM each of the forward and reverse primers (Generi-Biotech, Hradec Kralove, Czech Republic) (Table 1). Ten minutes initial heating at 95 °C followed 45 cycles at 95 °C for 15 s and 60 °C for 60 s. The mixtures were incubated and measured in duplicates on an iQ cycler with iQ5 Optical System Software 1.0 (Bio-Rad, Hercules, CA, USA). Cq for villin, claudin-1, claudin-2, and occludin were normalized to β-actin and cyclophilin A and their relative mRNA fold change expressions were calculated by 2^−ΔC_T method [24 (link)] by GenEx 6.1 software (MultiD Analyses AB, Gothenburg, Sweden).

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)].