We used the Renilla Luciferase Assay System from Promega (cat#E2810) and the Nano-Glo Dual-Luciferase Reporter Assay System (cat#N1610) according to the manufacturer's protocol. The luminescent signal was measured on a GLOMAX 20/20 luminometer. Statistical analysis was performed by one-way ANOVA with GraphPad PRISM 9.
For RNA and protein extraction from the luciferase reaction, 100 µL of TRI REAGENT-LS reagent were added to 100 µL of the luciferase reaction. Samples were stored at −80°C prior to processing according to the manufacturer's recommendations. RNA pellets were resuspended in 12 µL of FAE (formamide, 10 mM EDTA) (Shedlovskiy et al. 2017a (link)), and equal volumes of the dissolved RNA (5 µL) were analyzed by northern hybridizations using [32P]-labeled probes specific for 18S and 25S rRNAs. The radioactive signals corresponding to the rRNAs were measured as phosphorimaging units to obtain RLuc/18S rRNA and RLuc/25S rRNA ratios, where RLuc is the Renilla luciferase luminescence units, and 18S rRNA and 25S rRNA represent phosphorimaging units corresponding to the full length rRNAs in the same reaction.
For RNA and protein extraction from the luciferase reaction, 100 µL of TRI REAGENT-LS reagent were added to 100 µL of the luciferase reaction. Samples were stored at −80°C prior to processing according to the manufacturer's recommendations. RNA pellets were resuspended in 12 µL of FAE (formamide, 10 mM EDTA) (Shedlovskiy et al. 2017a (link)), and equal volumes of the dissolved RNA (5 µL) were analyzed by northern hybridizations using [32P]-labeled probes specific for 18S and 25S rRNAs. The radioactive signals corresponding to the rRNAs were measured as phosphorimaging units to obtain RLuc/18S rRNA and RLuc/25S rRNA ratios, where RLuc is the Renilla luciferase luminescence units, and 18S rRNA and 25S rRNA represent phosphorimaging units corresponding to the full length rRNAs in the same reaction.
