I am trying to isolate cfDNA from plasma samples (2-3 ml) for NGS. I get around 15 ng/ul concentrations of cfDNA from nanodrop but 10 times lower conc (1.4 ng/ul) from qubit dsDNA HS assay. Why is there such a difference and which one should I rely on? Is it possible to get an observably higher concentration of cfDNA from 4-5 ml plasma samples? I am using Qiagen's QIAmp Circulating Nucleic Acid Kit.
I always use Qubit for NGS application, and often had 10 times higher concentrations with Nanodrop with very diluted samples. However, Tapestation always confirmed the concentration of the Qubit, so I would stick with it if I were you. It could be that your concentration is simply too low for accurate Nanodrop measurement, which is usually not great at concentrations lower than 2ng/ul.
Hi,
The Qubit assay is in general more reliable, because it is based on fluorescence, but not absorbance as NanoDrop, which is very inaccurate for low concentrations of nucleic acids. I never worked with cfDNA, but when people bring RNA samples (for example), isolated with Qiazol, Nanodrop usually detects the phenol traces. And actual concentrations, measured with Qubit are much lower (10 times or more). So it could be that with NanoDrop you measure the traces of some aromatic compounds within your samples.
Anyway, 1 ng is already enough for DNA library prep with NexteraXT, for example.
Best,
Michail
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