Published in Volume
119, Issue 3 (March 2, 2009)
J Clin Invest.
2009;119(3):424–424.
doi:10.1172/JCI38802.
Copyright © 2009, American Society for Clinical
Investigation
Editorial
All data are not created equal
Ushma S. Neill, Executive Editor
Published March 2, 2009
An inauspicious start to 2009, unfortunately. This issue may seem a little thinner
than others we’ve recently published, as 4 articles that were previously
accepted and scheduled for publication in this issue will not appear. We continue to
screen all figures from accepted manuscripts, and we continue to find irregularities.
In several cases, the alterations in the figures led to the discovery of some
fundamental problems with the data. Many of the papers suffered from the same
problems, and this led us to consider whether it was time to revisit some
experimental basics.
Shall we start with some basic biochemistry? For the examples in this editorial,
let’s discuss Western blots in particular (though the rules apply to Northern,
Southern, and PCR blots too). We’ve stated before (1) that bands can be spliced together, but only, and I repeat
only, if they were noncontiguous but run on the same gel at the same
time. The figure then needs a thin line in between the spliced lanes and appropriate text
added to the figure legend to reflect the modification. Also, there is nothing more
reliable than a blot with bubbles — there is no need to erase background noise
or doublet bands.
I was always taught that a loading control, say β-actin or GAPDH, was probed
off the same gel. My understanding of a loading control is that it represents an analysis
of an irrelevant protein from the exact same gel lane to assess how much sample was loaded
in that particular lane. This seemed to be an elementary governing principle to me, which
was why I was somewhat shocked when we had an article that, among other problems, ran the
loading control on a separate gel at the same time. To me, running a parallel gel, even if
the sample run on the gel was an aliquot from the same tube, does not demonstrate equal
loading of sample in the experimental gel. How does one control for variations in pipetting
such small volumes? I realize that some blots need to be stripped and reprobed several
times, but can’t you just cut off the bottom and reprobe that part for the
actin or other loading control and reprobe the top for another protein of interest? Or
can’t you run duplicate blots and present 2 rows of loading controls? We are
worried about the manipulations we can detect, and I suspect that in cases such as these, a
heavy (or light) hand with a pipette can influence a band’s appearance when no
loading control is there to normalize it.
Another basic principle relates to exposure times. Specifically, in a particular row of a
Western, all bands presented should be from the same exposure time of the film. We allow
band splicing — with all the appropriate caveats, as described above. But
splicing lanes from various exposures of the same blot doesn’t prove anything.
If you expose most any gel long enough, you’ll get bands to appear and the
results you want. Space is not limiting, and you can always add another row of lanes to
your figure that show a longer exposure time to verify that the protein was there, albeit
in trace amounts.
This may seem elementary, but keep the raw data. A well-annotated lab
notebook can resolve problems very quickly. Scan the film from your blot and save it on
your computer and on the lab server, and keep a copy on a USB drive or elsewhere
(anecdotally, we once had an author claim he could not provide a high resolution version of
a Western blot from his accepted paper because all the data were on a single USB drive that
had been lost). Then, paste the film into your lab notebook. Label the film clearly and
annotate the date and conditions. And protect the lab notebook. Do not lose it when you
move your lab (another excuse from an author when he could not substantiate the data in his
accepted manuscript). Perhaps even e-mail a scan of the uncut, labeled film to yourself and
the senior author to ensure the data are accessible. And further than just saving
immunoblots — the same relates to histology and other data — print
them out. Make multiple copies. Label the data carefully. Keep clear records.
When questioned about data in a paper, many senior authors feign ignorance and blame the
first author or data generator, but I find that disingenuous. If you are the senior author,
it is incumbent on you to verify all of the raw data yourself. There is intense pressure to
produce, and to produce high-impact results. Sometimes this can lead to a student doing
anything to please. If the paper goes out with your name on it, you should be able to
verify every single piece of data in it and take responsibility for it.
We have tools to be able to detect whether you have altered your figures in any way. If you
cut it, crop it, squeeze it, tease it, or otherwise massage it — we can see it.
And you can be sure that we have closely examined all figures in the 19 other articles in
this issue. I hope that the experiments themselves in our published papers have been
performed properly, but that is not something we can police.
References
-
Neill, U., Turka, L.A. 2007. Navigating through the gray (and CMYK) areas of figure manipulation:
rules at the JCI. J. Clin. Invest. 117:2736.
