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?
1-on the 30:1 ratio, this is not exceptional (actually pretty usual): Renilla values are typically quite higher.
2- as to how to analyze your data I tend to normalize to within experiment values. In your case I would normalize, for each experiment, to WT. I would then average then average the inter experimental values. This means your Wt would always have a value of 1 and your MUT would (in this case) hover around 1. For the statistical significance, perform a 2-tailed t test. In your case, there might be a slightly (on average) lower MUT value, but it wouldn't reach statistical significance. You may want to repeat the experiment with different DNA preps and more repeats, to get more power and remove issues with DNA prep qualities etc...(particularly given the very small effect you are seeing).
Good luck!
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