Presumably one can use our CyQUANT Cell Proliferation assay reagent to measure the absolute DNA (or RNA) content per cell by a non-flow cytometric assay http://www.probes.com/media/pis/mp07026.pdf This reagent can measure cell number from 50 to 50,000 cells with total linearity and measures DNA + RNA. http://www.probes.com/handbook/figures/1504.html To get total DNA content only, treat with RNase. To get total RNA content only, treat with DNAse. The intensities are additive to get total nucleic acids in the sample (see figure in the first link). To get average DNA/cell in a known number of cells, lyse the cells, treat with RNase to get DNA only, add the CyQUANT GR reagent and measure the fluorescence. You will have to separately calibrate the fluorescence spectrometer (or microplate reader) with a known amount of DNA and the CyQUANT GR reagent to get a standard curve. Also, you will have to measure exactly how many cells were lysed by counting the cells, such as with a Coulter counter or flow cytometer. I expect that this will give a quite precise value since proteins, lipids and polysaccharides do not interfere with the CyQUANT assay. It is possible that this would work in a flow cytometer too after doing the assay once on a known suspension of the marine organisms but you would have to see if you can obtain a conversion factor that relates the absolute amount of DNA measured by the CyQUANT solution assay above with some intensity you measure using flow cytometry. Both the fluorometer and flow cytometer have arbitrary fluorescence units so unless you can relate you flow cytometric measurement back to some absolute measurement you cannot get absolute DNA per microorganism. What may allow you to do this with be measuring the DNA standard that is used to calibrate the CyQUANT fluorescence intensity by absorbance and the cell number by counting. It may be possible to count the cells in a sample with a flow cytometer (best using a bead standard mixed in, which will not interfere with the CyQUANT assay) then lyse these into the CyQUANT GR assay reagent as described above. If the sample only contains cells of interest and no other DNA-containing materials, you can use the CyQUANT assay to measure how many without any flow cytometry. The CyQUANT assay, of couse, was designed for measuring cell proliferation as an alternative to thymidine incorporation. Rob Wadley wrote: > Dear All, > > I have an Assoc. Prof. who is looking for any modern variant of a > technique popular in the 1970's. Or a good old method would be fine > too. > > In particular she wants to perform a 'C value' test to determine DNA > content as picograms/nucleus, by flow cytometry from marine organisms. > > I'm afraid the 1970's are well before my time in science I was still > building cars, houses & spinning wheels, but if anybody can help me > out? > > Regards > > Rob W. > > Robert Wadley, Laboratory Manager, > Cellular Analysis Facility > University of New South Wales, Sydney, > Australia, 2052. > > Services available 24 hrs/7 days > Consultancy service available > > Ph: +61 (2) 9385 3517 > Fax: +61 (2) 9385 1591 > Mobile: 0411 874 470 > E-mail: r.wadley@unsw.edu.au > E-mail: rbwadley@hotmail.com > WWW: http://www.micro.unsw.edu.au/ > (Centres, CAF)
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