A few days ago I posted about a problem we're having interpreting some microarray data. That problem could be simply resolved by finding the missing notebook, or activating the relevant memory synapses in the brain of the student who did the work. But there's a bigger and more intrinsic problem with this type of experiment, and that's deciding whether observed differences are due to antibiotic effects or to chance.
Consider data from a single array slide, comparing the amounts of mRNA from each of ~1700 genes from antibiotic-treated and untreated cells. If the antibiotic has no effect on a gene we expect the amounts from treated and untreated cells to be 'similar'. We don't expect them to be exactly the same (a ratio of exactly 1.00) because all sorts of factors generate random variation in signal intensity. So we need to spell out criteria that let us distinguish differences due to these chance factors from differences caused by effects of the antibiotic treatment. Here are some (better and worse) ways to do this:
1. The simplest way is to specify a threshold for significance, for example declaring that only ratios larger than 2.0 (or smaller than 0.5) will be considered as significant (i.e. not due to chance). But this isn't a good way to decide.
2. We could instead use the standard statistical cutoff, declaring that only the 5% highest and 5% lowest values would be considered significant. One problem here is that this criterion would tell us that some differences were significant (5% of 1700 x 2 =170) even if in fact the antibiotic treatment had absolutely no effect.
3. We could improve this by doing a control experiment to see how big we expect chance effects to be. One simple control is to use a microarray where both RNAs are from the same treatment. We can then use a scatter plot to compare the scores for each point. The diagonal line then represents the 1.00 ratio expected in the absence of random factors, and the degree of scatter of the ratios away from 1.00 tells us how big our chance effects typically are.
We could then decide to consider as significant only effects that are bigger than anything seen in the control. Sometimes this could be easy. For example, if expression of one gene was 20-fold higher after antibiotic treatment, whereas control effects never exceeded 5-fold, we'd be pretty sure the 20-fold increase was caused by the antibiotic.
But what if the control effects were all below 2-fold, and two of the genes in our treated RNA sample were up 2.5-fold? Does this mean the antibiotic caused this increase? How to decide?
We need to do enough replicates that the probability of any gene's expression being above our cutoff just by chance is very small. For example, we could require that a gene be expressed above the 2-fold cutoff in each of four independent replicates. Even if our arrays had a lot of random variation, comparing replicates can find the significant effects. Say that our controls tell us that 10% of the genes are likely to score above the 2-fold cutoff just by chance, and we do see about 170 scoring that high in our first real experimental array (comparing treated and non-treated cells). If we do another experimental array, we again expect 10% to score above the cutoff, but if only chance determines score, only about 17 of these are expected to have also scored this high in the first replicate. If we do a third replicate, we only expect 1 or 2 of the genes scoring above 2-fold to have scored this high in both of the first two replicates. You see the pattern. So, if our real four replicates include 6 genes that scored above 2 in all four, we can be quite confident that this is not due to chance. Instead the antibiotic treatment must have caused their expression to be increased.
The real analysis is more complicated, in part because different replicates have different amounts of error, and in part because we might want to take into account the actual expression levels rather than using the simplistic cutoff of 2-fold. Our preliminary look at the real replicate data last week suggested that a few genes were consistently (well, semi-consistently) increased. Tomorrow we're going to take another look at the real data and see if we think this is a result we have confidence in.
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