Hi,

I'm looking for some advice in analyzing my luciferase data.

I study the effect of mutations in 5’-UTR on translation efficiency. For this I’ve cloned 5'UTR of the target gene (without (WT) and with (MUT) studied mutation) to psiCHECK-2 vector before Renilla Luciferase CDS. I transfected WT or MUT plasmid into HEK293T cells separately. 48h after transfection I used Dual-Glo® Luciferase Assay to detect Firefly and Renilla Luciferase activities. For each plasmid I have 3 technical repeats (different wells, same transfection reagent master mix) and 3 biological repeats.

I’ve done Renilla/Firefly normalization in each well and obtained 9 values for each sample.

The obtained Rluc/Fluc ratios in different experiments are presented in the table below.

I have 2 questons:

1. My raw Renilla signal is about 30 times lower than Firefly, so I have low value of Rluc/Fluc ratio (about 0.03). Is it suitable or is it a sign of measurement problems?

2. I have the same MUT/WT ratio in all experiments, but initial values of Rluc/Fluc have a great variability. How should I statistically analyze this data in a best way? How to calculate mean and SD value for biological replicates?

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