Need help on "Dilution method" - "Dilution method" (May/12/2008 )
Dear every one,
I try to measure DNA level in the plasma without purification as desbribed in the following paper: "The Use of Fluorometric Assays to Assess the Immune Response to DNA in Murine Systemic Lupus Erythematosus, PMID: 12791090".
These authors have been using this method for years and just desbribed briefly as following:
"Assay of plasma DNA. Plasma DNA was assessed by a fluorometric assay. For this purpose, diluted plasma was
mixed at a 1 : 1 ratio with the dye PicoGreen (Molecular Probes Inc., Eugene, OR, USA) diluted 1 : 200 in TEN
buffer (10mM Tris, 1mM EDTA, 100mM NaCl, pH 8.8) in a black 96-well microtitre plate (Costar, Corning Incorporated, Corning, NY, USA). This buffer was used to minimize the nonspecific binding of DNA to other proteins
in the plasmas or sera . Fluorescence was measured using a TECAN GENios microplate fluorescence reader
(Salzburg, Austria), with an excitation wavelength at 485 nm and emission wavelength at 535 nm. Assays were
performed in triplicate for each sample. The DNA concentrations in plasma were calculated according to a standard
curve made from double-stranded calf thymus DNA (CT dsDNA)."
As you know the standard curve is linear, but the 2-fold serial diluted samples are not linear because plasma has some intrinsic fluorescence, and/or some effect on the fluorescence. I dont know how the authors can calculate the concentration of DNA.
I also sent an email to ask the authors but no response. I greatly appreciate if you could give me any advice and any paper doing the same dilution method.
The authors clearly did not worry about this, and perhaps you should not either. You could treat some plasma with DNAse to get rid of DNA (I'd probably add some RNAse as well). Then you could do a standard curve in the presence of this plasma and the buffers you are using. If the results are significantly different from their standard curve, then they have a problem. If not, then use their method. It all boils down to whether the intrinsic fluorescence and binding effects are significant. Find out.
the authors diluted the plasma. maybe this attenuates the intrinsic fluorescence?
thanks a lot for your suggestion.