Nardon C, Weiss M, Vieira C, Biémont C. 2003. Variation of the genome size estimate with environmental conditions in Drosophila melanogaster. Cytometry Part A 55A: 43-49.
These authors examined the effects of various temperatures and humidities during development on measures of genome size by flow cytometry in Drosophila melanogaster male adult brains. They also looked at the effects of varying temperatures of prepared solutions after extraction of cell nuclei, and of the age of the flies when they were killed.
Previous studies by various authors have found a range of factors that can affect genome size estimation, such as age of the individual or temperature. The results reported here are consistent with those earlier reports.
Unfortunately, I am rather suspicious of these results because of some odd phrases in this paper. There are some probably trivial and meaningless mistakes, such as refering to eclosion of adult flies from pupae as “hatching”, but other putative mistakes are more worrisome, such as the confusing discussion of the statistical tests employed. Five of seven experiments in temperature (i.e. five of seven comparisons between 17°C and 25°C) are reported to show differences in measured stain flourescence intensity, but the next clause says that three of those five were significant differences. If it is not significant, it is not different, no?
Their data seem to show some noise, which is of course to be expected in any experiment. They interpret this noise in a somewhat confusing manner, never refering to it as “noise”. I am not certain I understood the discussion section properly.
The most important finding in this paper to me is probably the warning that prepared samples of extracted nuclei must be maintained at low temperatures (i.e. on ice) for consistent results.
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We had a pretty detailed discussion about this one in lab meeting, but it must have been before you arrived.
Thanks for commenting!
I'm not surprised a lab meeting had a discussion about this paper - there's lots here to think about. I'll try to bring it up with one or two people in the lab and get some impressions.
Have a look at their PI concentration and stain time, to start with.
Ah, yes, thanks. Nardon et al. (2003) used 1ug/mL of Propidium Iodide, whereas Tiersch et al. (1989) used 25ug in 0.5mL. Nardon et al. (2003) incubated on ice for 10 minutes; Tiersch et al. (1989) don't state directly their incubation time; I don't have either of the references they cite in that part of their Methods. I've been told to use 20 minutes minimum by experienced Flow Cytometry users.
I just read their more recent paper (Nardon et al. 2005). As I wrote in my entry (before reading the comments here), the methods in 2005 are very similar to the methods in 2003. I shall have to devote some reading time to the details of Flow Cytometry methodology, along with some discussion with other people. I don't yet understand how important certain methodological differences might be between studies.
Um, well, you could start with this one:
DeSalle, R., T.R. Gregory, and J.S. Johnston. 2005. Preparation of samples for comparative studies of arthropod chromosomes: visualization, in situ hybridization, and genome size estimation. Methods in Enzymology 395: 460-488.
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