You're right that one should focus on the variability of interest; but the presence of a stronger variability in another dimension can ruin your experiment. Consider, for example, you want to see how the left bit of the liver is different from the right. You take one sample of each side, and spend all the time and money to finish RNA-Seq and differential expression, and get a gene list with fold-change. You don't know yet if this holds for a second animal, or even if your samples were of a similar material. What if oopsy your left bit of liver had a vein running through it, and that's really the source of the difference seen. You dont know if the difference is really the liver gradient you wanted to measure, or just a sample problem. This can be remedied by taking two samples at each point. Then the same argument applies to the whole animal. They are much more different than you know. Really have to do two or three whole separate animals, and maybe then one bit of left and one bit of right liver will capture the real diversity present. Until you do the measure you don't know.
If you were interested in technical variability, then you should measure the same bit of tissue twice. Here the problem is possibly the variability is present in a gene that is not being transcribed in that animal, so without a biological replicate, you have underestimated the technical variation.
It's important to think about the possible sources of variation when designing the experiment. If money was free, we should always do more samples from more independent animals. When money is limited, we have to choose carefully how many samples of what kind are needed.
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karl.stamm
You should think long and hard about what exactly you want to get out from an experiment that homogenizes a whole animal and looks at the RNA..
It is true, but for small organisms (e.g. Drosophila) it may be hard to dissect tissues and extract RNA, specially for large sample sizes.
On the other hand, you can view these whole organisms extractions as an "holistic" approach. ;-)
Given that RNA expression is tissue specific the 'holistic' approach is likely to wash out any signal. If you're unprepared to do something difficult properly then don't do it at all.
Well, there are times when it's the right thing to do - like as h.mon mentioned Drosophila are often ground up and sequenced because you are looking for global changes, or extracting the cell is a pain, or you dont know the cell type affected yet (give high-methyl diets to flies and see what gets turned on/off). But certainly you have to be mindful of diluting out signal. I think as long as you respect that, its OK.
Your statement is very wide and bold, so wide and bold it is likely to wash out any signal. ;-)
My comment about "holistic" was more a joke than serious, it is about spinning a shortcoming into an advantage.
Anyway, in an ideal world we really would like to have a finer-grained picture considering tissue, spacial location of cell on said tissue, type of cell within tissue, etc. However, there are several constraints on what we can do - the most obvious being time and money, but also number of skilled people working, difficulties in obtaining samples, and so on. And as John said on his comment, there are experiments where effects are global (see here and here for two papers I like), and dissecting is a pain.
I fundamentially disagree. I note that the second paper you mention is one of mine and is very different to what you're proposing.
In our A. thaliana study we only took wild-type two-week old seedlings which are early in development with little variability and few tissues. We obversed what the global transcriptome structure looked like in terms of 3' end formation. We made no assessment of differential gene expression.
Likewise in the other experiment only the sex chromosomes were observed which are of course exquisitely tissue specific, so taking whole organisms is probably ok.
I disagree fundamentally because if an experiment is too hard or expensive do to properly then it shouldn't be performed. You'll save yourself and everyone else a lot of wasted time chasing down false positive data. If you're interested in tissues-specific gene expression differences then look at the tissues not the whole animal.