Dual Luciferase assay data analysis issues
#1
Posted 28 August 2012  11:50 AM
I want to investigate the functional properties of a promoter fragment of interest. I cloned the promoter into a pGL3 plasmid and co transfesfected this promoter plasmid with a renilla vector in HEk293T cells to assess transfection efficiency. After following the protocol for the DualGlo luciferase assay system and assaying luminescence, I normalized the data to empty vector control as recommended by Promega.
Just looking at the data, it seems that there is a significant difference in my experimental promoter fragment compared to empty vector controls. The luciferase normalized ratios are on the order of 1.7 compared to empty vector at 0.7. and an even greater difference when calculating relative response ratios. However doing a 2tailed student's T test shows that it is just under statistical significance. I recently read a paper (Zhuang, H et al 2009) in Nature Protocols that shows a slightly different method of analysis. When I do this analysis, I get statistical significance.
Please help!! I'm not sure which one is best or better represents my data since I get two different results. Does anyone know a good way to analyze/normalize the luciferase values compared to emtpy controls?
#2
Posted 28 August 2012  01:47 PM
If you answered no to either of these, then a Student's ttest is not the correct test to be performing, as it is only for normally distributed data for large sample numbers. The equivalent, slightly less powerful, but more statistically valid tests are known as nonparametric tests, they are for low sample numbers and/or nonnormally distributed data. The tests you probably want to perform are either the MannWhitney test (for unpaired groups) or the Wilcoxon test (paired groups). KruskalWallis is the equivalent to ANOVA if you need it.
#3
Posted 28 August 2012  02:30 PM
Andreea
#4
Posted 29 August 2012  07:56 AM
Bob, you are correct, I have a small sample size and cannot assume a normal distribution curve. I will try the calculations with different tests and see how the data looks.You are right as well Andreea; I have seen numerous papers on pubmed using a student's ttest with few samples, so that is where some of the confusion occured.
I actually subcloned the promoter plasmid into eighths so I essentially am testing the activity of 7 smaller promoter fragments, the full promoter fragment, an empty vector control, and mock transfected cells in triplicate. So essentially, my sample size is 10. One thing that I have notcied is that, most papers don't give statistical significance and rather just mention a fold change or relative luciferase activity. So would these tests hold true in this scenario as well or should I just stick with the "norm" and comment on a fold change?
#5
Posted 29 August 2012  01:39 PM
If you can generate a standard deviation on your data and plot it, basically if the standard deviations overlap, then there is no statistical significance.
Actually, you have 10 separate groups of samples, each of which I would expect to have a normal distribution of the measured luciferase values, due to the nature of the way these sorts of experiments are measured. You need to be using an ANOVA (KruskalWallis) style approach to analyse your data, or you run the risk of hitting a type one statistical error. You can do posthoc testing (e.g. Tukey test) to find out which group(s) are statistically different amongst the groups.I actually subcloned the promoter plasmid into eighths so I essentially am testing the activity of 7 smaller promoter fragments, the full promoter fragment, an empty vector control, and mock transfected cells in triplicate. So essentially, my sample size is 10. One thing that I have notcied is that, most papers don't give statistical significance and rather just mention a fold change or relative luciferase activity. So would these tests hold true in this scenario as well or should I just stick with the "norm" and comment on a fold change?
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