...the one where the treatment is applied once to each animal or cell culture, and then the effects (phenotype, gene expression...) are analysed after different time-points (2, 5, 7 days) to be compared mutually,
or
... the treatment is applied at different time points such that the effects are analysed simultaneously,,so the time passed between the treatment and the analysis is the same as in the first case (2,5,7 days-treated objects).
The former is in my opinion better from a technical and practical standpoint. It's easier to set up, to plan, and to carry out. Collect samples as you go along, freeze them.
If you're worried about possible non-treatment related ageing effects or something, just include adequate control groups so that each timepoint has treated and untreated samples.
The only reason I can think of for using a method like the latter is if samples needed to be analysed quickly using an expensive technique that is cheaper or more practical to use on all samples at once. Radioactive transport assays using the "centrifuge stop" technique, for instance (though there we're talking minutes not days).
I am actually more concerned for the variability of the readouts, more than the variability of the treatment, which is usually a simple preparation used in a single concentration. The experimental readouts are to be collected immediately and are time-consuming, complex (such as multiple phenotype analysis, follow-up co-culture assays, and so on) and, in the former case, should be analysed as independent experiments (i.e. index of inner controls).
I think that first approach is more accurate. Better to have controls also for all time points as time course is long. If time intervals are smaller like in min or in hours, having controls for each time point is not important
Thank you all for the answers. As I understand, all of you prefer the first approach, and this one is the most common. I wonder, if there is any crucial mistake of the second approach, considering that the readouts may vary in a larger extent that the treatments (the same non-treated controls could be used in the second approach as well)?
Sergej,
The first approach is more appropriate regarding the quality of the experiment, especially in biomedical research.
However, if your analytical methods are very complex, not available in-house, need expensive equipment/large team to execute, the second approach may be considered.
Dear professor Pelicon,
I much appreciate your input on this matter and it's very nice to hear it from you. I myself would choose the first approach if there were no complexity in the experiment readout. However, I still looking for any mistakes in the experiment set the other way around. The problems might come from variability of the treatment for sure, but they might come from the readouts as well. Therefore, I am still weighting those two.
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