Cfx connect real time pcr rt pcr detection system

Gene expression levels were analyzed using the CFX Connect Real-time PCR (RT-PCR) Detection System (Bio-Rad, Hercules, CA, United States). RT-PCR was performed in duplicate using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, catalog no. 1725274). The data were analyzed using Bio-Rad CFX Maestro 1.0 software. The specificity of the reactions was determined based on dissociation curve analysis. Fold changes were calculated using the ΔΔCq method as described before (Majewski et al., 2019 (link)). Expression levels were compared between groups using analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test. The 18S ribosomal gene was used as a reference. The sets of primers that were used in the analysis are shown in Table 1.

For FRET-melting experiments, FAM-AT11-L0-TAMRA was used at a concentration of 0.2 μM, and the ligand concentrations used were 0.2 μM (1 molar equivalent), 0.4 μM (2 molar equivalents), 0.6 μM (3 molar equivalents), and 1 μM (5 molar equivalents). The oligonucleotide was annealed as described before in 20 mM potassium buffer (pH 6.9) supplemented with 65 mM KCl. FRET-melting competition studies were performed with 3 μM (15 molar equivalents) or 10 μM (50 molar equivalents) of ds26 (a double-stranded sequence) in the presence of FAM-AT11-L0-TAMRA at 0.2 μM and the ligands at 0.6 μM (3 molar equivalents). Thereafter, the samples were incubated for 30 min at room temperature. After 30 min incubation, samples were incubated on a thermocycler (CFX Connect™ Real-Time PCR (RT-PCR) Detection System (Bio-Rad, Hercules, CA, USA), supplied with a FAM filter (λ_ex = 492 nm; λ_em = 516 nm) at 25 °C during 5 min followed by temperature increases of 1 °C at each 1 min, from 25 °C until 95 °C. FAM emission was measured each time the temperature was increased. Each condition was tested in duplicate on three independent 96-well RT-PCR plates. Data curves were normalized to calculate the melting temperatures. To assess the competition effect, the Selectivity factor (S factor) was determined following Equation (2).
$$\mathrm{S} \mathrm{factor}=\frac{\mathsf{\Delta }{T}_{\mathrm{m}}\mathrm{in} \mathrm{presence} \mathrm{of} \mathrm{the} \mathrm{competitor}}{\mathsf{\Delta }{T}_{\mathrm{m}}\mathrm{without} \mathrm{the} \mathrm{competitor}}$$