Hi
I'm looking for some help in analysing my luciferase data. I have a transcription factor and then a range of proteins I think will influence transcription factor activity.
For each condition I have 5 technical repeats (different wells, same transfection reagent master mix) and I have done this 3 times (so I have 15 values in total).
For each reading I have taken luciferase and renilla readings. I also have background readings (untransfected cells) for both luciferase and renilla.
How am I best to analyse the data?
So far I have done luciferase/ renilla and have then subtracted the average reading for the background (i.e. background luciferase/ background renilla). I have excluded any values that fall below this background reading. I have then performed the grubbs test on the 5 technical repeats to exclude any outliers and then taken an average so I end up with 3 values. Do I perform statistics on these three values or do I perform on the full 15 values? Also if I wish to express as % of control, do I do the data analysis on the %s?
I'm having a bit of trouble as my three repeats have such varied responses. For instance, some of my fold changes are as shown:
100% 100% 100% control
66% 153% 123% protein 1
74% 232% 142% protein 2
88% 180% 81% protein 3
Thanks for any help you can offer!
Tara, have you normalized your fluorescence signal to total protein? That may reduce variance of your biological replicates.
Is your transfection efficiency similar between the replicates? Check the signal of controls.
Is your control positive (e.i. your transcription factor is active under the conditions and the protein treatments lower the transcription) or negative (transcription gets activated only after the protein treatments)? We use cell line with stably transfected luciferase reporter and yet the basal transcription activity (untreated control) varies between the experiments. But our signal is strong enough to see that the treatments increase luciferase transcription.
Vojta
Hi Vojta many thanks for your reply.
I was previously normalising to protein but due to some of the treatments causing cell death I had too low protein concentration to appropriately quantify.
I have included a positive control (a known agonist of the TF) and this gives the FC 184% 509% and 316% so there is variation between the experiments.
The transcription factor is active under control conditions and I'm hoping to see an increase or decrease when protein is included
Many thanks
Tara, if the treatment kills the cells, I wouldn't believe the results of the assay. Is that physiologically relevant? Unless you are studying the effect of the treatments in the apoptotic cells...
How about testing concentration series of your compounds to see if the effects are specific and concentration-dependent? Are you limited to small cultivation format and so you do not use larger flasks to get more cells for accurate protein concentration determination?
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