Why the he..... is the results so differnet? - Picogreen vs. nanodrop DNA quantification of buccal swabs (Jun/08/2007 )
Hi,
I got this strange problem... I have recently extracted several buccal swabs and needs to do some DNA quantification. We have two systems in our lab, a nanodrop and PicoGreen... When I quantify the DNA by picogreen we get, app. 1-2 ng/ul, thats relatively low... when we do nanodrop we get 50 ng/ul... thats a big differnece.. I know that pico only measures dsDNA, but seriously... isent that a big differnece?? Has anyone had the same experience or a god suggestion on why I see this differnce??
Thanks!
we have seen this in our lab as well, in our case it turned out the extraction protocol was not compatible with picogreen so we just used the nanodrop. you could possibly test experimentally which concentration is right on a gel or something, I don't remember why it is incompatible, just said so in the protocol...
HTH
Cool, great to hear that I am not a raving lab maniac! 
I will try to run it on a gel instead, thank for your response to my question, I really appreciate it!
I will try to run it on a gel instead, thank for your response to my question, I really appreciate it!
We observed the same while comparing Nanodrop with qdot (invitrogen). Out technician then tested both with standardized solutions. It seems as if the Nanodrop in general gives higher concentrations. If you want I can ask her what was the exact outcome of this and what the companies had to say about it.
This is a problem for us too. our lab has a Nanodrop ND8000 and we isolate genomic DNA with the Roche MagNA Pure LC blood and tissue systems. We measure the purified DNA and ship it to our collaborators. The problem is that prior to their microchip analysis they use the picogreen system and generally measure lower concentrations than what we do. In general they measure approx 50% of what we do on the nanodrop. they claim that picogreen gives more exact DNA concentrations, but is it necessarily like that? OK, picogreen measures dsDNA, but is that method more accurate than nanodrop? Is the method that gives the lowest concentrations always to be considered as the best method? Two different systems would necessarily have their strengths and weaknesses, right?
Even plain dNTPs will give a concentration on the nanodrop; I suspect the difference is that the nanodrop is measuring everything in solution, including left over protein and RNA etc, whereas picogreen is specific for double stranded nucleic acids.
But still the 260/280 ratio is 1,9-2,1 which should indicate no or very little protein contamination (?). It could be RNA since the roche protocol does not have any RNase digestion step, but is it that much contaminating RNA compared to genomic DNA? we're talking mass and not mole... Another thing, in the nanodrop software you can choose between DNA and RNA concentration measurements, is that just a gimmick?
The only difference between RNA and DNA is an oxygen. There is essentially no difference between the absorbances of RNA and DNA. The equations used by the nanodrop for calculating RNA and DNA concentrations only differ in that the DNA uses 40 and the RNA uses 50 as a multiplier.
I have always used Qiagens QiaAmp columns for extraction. I found that with their QIAsafe products I was able to store DNA no problem. I think that might help!