DNA comparison between chicken and trout standards

matt parrow mwparrow at unity.ncsu.edu
Mon Jul 9 08:26:19 EST 2001

	 The Chicken Erythrocyte Nuclei (CEN) and Trout Erythrocyte Nuclei (TEN)
standards I purchased from Biosure Corp. exhibit very nice DNA peaks with
excellent CV's on my instrument.  Each is also very consistent internally in
DNA fluorescence.  However, the two standards do not seem to compare with
one another in my preparations.  I would expect the trout (published ~5 pg
DNA per 2C nucleus) to exhibit roughly twice the fluorescence as the chicken
(published ~2.5 pg DNA per 2C nucleus)when both are stained and analyzed
	Regarding the comparison between the 2C CEN and TEN DNA content: the known
2C DNA content of chicken is published as 2.33 pg DNA per nucleus (it is
also published as 2.5 pg).  The published value for rainbow trout is
published 2C = 5.17 pg DNA per nucleus.  Thus the 2C TEN / CEN DNA ratio is
5.17 / 2.33 = 2.21  .  However, when both CENs and TENs are DNA stained
similarly and analyzed by flow cytometry, the mean peak value ratio for 2C
TEN/CEN is nowhere near 2.21, at least in my preparations and on my
instrument (Coulter EPICS ALTRA).  The fluorescence peak ratio I measure is
closer to 4.1  .  Can anyone suggest why?  I realize that different trout
broodstocks and chicken lines may have slightly differing DNA contents, but
I am not aware of strain-specific differences of this magnitude.  Thus I am
unable to satisfactorily resolve this issue.  I am using a DNA fluorochrome
(SYTOX Green) that does not exhibit base-pair specificity, and I have
checked the linearity of the signal amplification system using fluorescent
beads - it appears fine.
	In the excellent paper by Vindelov et al. in 1983 (Standardization of flow
cytometric DNA analysis by use of chicken and trout rbc's as reference
standards), the authors use both chicken and trout rbc's and "corrected the
TRBC:CRBC ratio by changing the zero-point so that the ratio for each
histogram was equal to 2.28 .  Can anyone tell me how this is done (i.e what
exactly is the parameter one adjusts to "change the zero point" such that a
biologically correct DNA peak fluorescence ratio can be achieved between
these two standards?
	I am relatively new to flow cytometry, and it is quite possible that I am
simply missing some fundamental aspect in this standards comparison.  Any
advice or information anyone can offer is greatly appreciated.

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