Saturday, September 10, 2016

Relative Fluorescent Units Considered Harmful

My working title for the paper that will come from this year’s iGEM interlab study is “Relative Fluorescent Units Considered Harmful.”  It’s a bit of a playful title, invoking a computer science tradition started by the notorious Edsger Dijkstra. I think, however, that this statement is deserved and also that we can now back it up with some hard experimental evidence.

Most of the data is in—I’m just waiting for a few more teams with extensions—and it looks like we’ve got amazing results.  The big news is, in a more positive reformulation of my title, that calibrating fluorescence measurements works, and that it makes a big enough difference to be worth it.  Let me present the key conclusions that I believe we can now support in the form of responses to the most common arguments that I hear against calibrating fluorescent measurements.
Q: Wouldn’t it be difficult and costly to add fluorescence calibration to experiments?
A: The materials needed are quite inexpensive. As for difficulty: it seems to be pretty easy for undergraduates and high-school students all around the world, so professional researchers should be fine.
Q: Aren’t calibrated measurements pointless, because cell behavior varies so much in different people’s hands?
A: Not according to our results: pretty much everybody who got the protocols right, as indicated by reasonable control values, had a tight correspondence in the rest of their values as well.

Q: Aren’t arbitrary or relative units good enough, if we just want to compare fluorescence?
A: Absolutely not! You know what I said about getting reasonable control values? In our study, anybody whose controls were wonky appears to have been a lot more likely to have wonky results elsewhere too, probably indicating some sort of protocol failure.  With relative values, however, a lot of those apparent protocol failures go through, polluting the data and potentially making all sorts of trouble down the road.
Q: Why can’t we just compare to a known system in a cell?
A: This is the idea behind Relative Promoter Units (RPU) and the like, which are pretty clever.  Just as with purely arbitrary units, however, if something goes wrong in the protocol, it’s likely to affect the controls as well: RPU also appears unlikely to have caught a lot of the problems that absolute units identify in our study, again leading to pollution of data with all sort of strange failure modes.
In short, calibrated fluorescent units make a big and quantifiable difference and they’re easy to use.  Moreover, given what we’ve seen, I suspect that a lot of the “cells are so touchy and behave so differently” laboratory folk-wisdom out there is really not about the cells, but about problems with culturing and measurement protocols that go unnoticed when you’re using relative or arbitrary units.

I wish that I could say more now about what we’ve learned, but we’re planning to announce the full results at the iGEM Jamboree at the end of October, and I’m embargoing all of the details until that time.
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