Microtiter plate based dual luciferase protocol

Majority of stop codon readthrough assays in this study were performed using a standard bicistronic reporter construct bearing a Renilla luciferase gene followed by an in-frame firefly luciferase gene, originally developed by (52 (link)). Separating the two genes is either a tetranucleotide termination signal (UGA-C) or, for control purposes, the CAA sense codon followed by cytosine. In indicated cases, the termination signal and/or the following nucleotide context was modified. It is noteworthy that this system avoids possible artifacts connected to the changes in the efficiency of translation initiation associated with the nonsense mediated decay (NMD) pathway (53 (link)), because both Renilla and firefly enzymes initiate translation from the same AUG codon. For further details please see (54 (link),55 (link)). All experiments and data analyses were carried out according to the Microtiter plate-based dual luciferase protocol developed by (56 (link)) and commercially distributed by Promega. Readthrough values are represented as mean ± SD from triplicates (n = 6) and each experiment was repeated at least three times. The raw data for all dual luciferase assays (absolute luciferase activities) are given in the Supplementary Excel File.

The majority of stop codon readthrough assays in this study were performed using a standard bicistronic reporter construct bearing a Renilla luciferase gene followed by an in-frame firefly luciferase gene. Separating the two genes is either a tetranucleotide termination signal (UGA-C) or, for control purposes, the CAA sense codon followed by cytosine. In indicated cases the termination signal and/or the following nucleotide context was modified. It is noteworthy that this system avoids possible artifacts connected to the changes in the efficiency of translation initiation associated with the NMD pathway (Muhlrad and Parker 1999 (link)), because both Renilla and firefly enzymes initiate translation from the same AUG codon. For further details, see Keeling et al. (2004) (link). All experiments and data analysis were carried out according to the Microtiter plate-based dual luciferase protocol developed by Merritt et al. (2010) (link) and commercially distributed by Promega. Readthrough values are represented as mean ± SD from quintuplicates (n = 5) and each experiment was repeated at least three times.