I work with porcine samples, unfortunately we only have 5 animals/treatments. Is it enough for a proteomics experiment, do I have sufficient technical reps?
Please provide more information.
Is your experiment only Western blots?
Are you identifying proteins or post-translational modifications (PTMs)?
Are you doing qualitative mass spectrometry (MS) or quantitative MS?
While the bare minimum for statistical analysis would be n=3 with one replicate, this is typically unacceptable for publication. n=5 with one replicate would suffice, but it does depend on the type of proteomic analysis being performed.
its LC/MS experiment.....however I read some papers which mentions to have atleast 6 technical replicates
http://www.ncbi.nlm.nih.gov/pubmed/18412383
We have published proteomics experiments with 3 biological replicates of a given physiological condition. In our hands from each biological replicate we can only perform one technical replicate ! The proteins extracted from each biological replicate were digested and the resulting petides injected in nanoLC for further off-line MS-MS analysis. We explained that a result was considered as positive if a given protein was identified in at least two injections out of the three.
We usually got the same problem. What we do is to use one sample or two and perform some technical replicates to check if everyting its working properly. Then I agree with Yannis, as long as you put some presence conditions for your proteins, 5 biological replicates will do fine.
The best way to review a biological result with less replicates is to repeat the experiment. Otherwise your proposition has no strength! I Know the problems with animal experiments, but if you want to do respectable science you have to do it.
Proteomics experiments often have the problem of repeatable results, because of the data acquisition methods (stochastic effects of mass exclution ec..). The only way here ist to increase the sample volume.
However good luck
Christian
originally posted by Sridhar Radhakrishnan
"its LC/MS experiment.....however I read some papers which mentions to have atleast 6 technical replicates"
In your initial post you mentioned having 5 biological replicates (5 different animals) per treatment, but nothing about technical replicates. A technical replicate would be repeating the same procedure/experiment with sample from the same animal (or an aliqout of the exact same sample from the same animal).
Biological replicates control for naturally occurring variation within a population (that is, variation that is just a natural consequence of inherent genetic or physiologic variation within the population).
Technical replicates control for variation due to vagaries in experimental procedure or protocol or instrumentation error.
The two terms refer to controls for very different sources of variation and should not be confused with one another.
I am sorry......my question was not clear.......I understand from above answers that 5 bio reps are fine..........actually I wanted to transition next to no of tech reps.........sorry that I did a horrible job of transitioning to next question........
However, I understand from responses above that multiple injections are fine as long as I detect presence of protein from at least 2 of 3 injections...
Sridhar
It depends of the objective of your study. For a discovery experiment 5 biological replicates could be ok, but to validate the findings you´d need to increase the number of individuals. For quantitative analysis, it´s also good to perform a power analysis in order to have an idea of the correct number of replicates you will need.
Cheers
Ignacio
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