A mystery on DNA content assessed through PI staining

Dear colleagues,
I have been helping a young PhD student from Lisbon to set a procedure for
measuring DNA content by propidum iodide staining in several species of
fish. Based on papers already published, he has followed a method that now
is leading to data that somehow contradict previous results. I told him
about this emailing list of experts on flow cytometry and ask him to
address you aiming to get helpful advice, as I’m not so much aware of
subtleties that could affect DNA staining techniques.
Thanks a lot in advance,

Alexis Pérez, PhD
Cell Imaging Unit
Instituto Gulbenkian de Ciencia
Portugal


Dear All,

I’ve been analysing fish blood samples using Flow Cytometry to quantify
nuclear DNA
content. I’ve followed published procedures, although so far I’ve been
unable to
replicate published results.
In the published paper they used blood collected from the caudal vein of
fish,
stored in freezing solution(*) at –80ºC, following Dawley & Goddard’s
(1988) procedure.
Samples were thawed and stained with PI solution (**) (30uL blood
sample+300uL PI
solution) for at least 10 minutes and analysed in Epics Profile II Cytometer
(Coulter) with chicken blood (standard) treated the same way. Assuming
that chicken
DNA content is 2.5, they calculated the DNA content of the species I’m
interested in
ranges from 2.7pg to 3.8 pg (only one species exhibiting 2.7pg, the others
between
3.7-3.8pg).
When trying to replicate their results (using the same cytometer and a
FACScan), I
obtained consistent, but conflicting results. Using the same value of
2.5pg for
chicken, my samples vary between 3.4-3.6pg. Readings show clean peaks and
CVs are
very low (2-3). Also, I stained my samples for 1 hour, increasing the
likelihood
that PI stained all DNA in each cell. I noticed that very concentrated
samples
yielded nice peaks with lower values than when they were further diluted.
Finally, I must mention that the “2.7pg” result was unexpected and the
authors
repeated their readings (5 specimens total for that species), getting the
same
results. This fact gave them confidence on the readings.
I would much appreciate if you could help me solve this “mystery”. Thanks
a lot!

*Freezing Solution (40mM citric trisodium salt, .25M sucrose, 5% DMSO, pH
7.6)
**PI Solution (500mL - 500 mg citric trisodium salt, 500 uL Nonidet P40,
25 mg Propidium Iodide, 25 mg RNase A, pH 7.6)

Hugo Gante,
University of Lisbon
Portugal