Should I use Nanodrop or Qubit? The DNA quantification dilemma.
|Qubit Fluorometer||NanoDrop ND-1000|
|Quantification method||Fluorescence-based dyes that bind specifically to DNA, RNA, or protein||UV absorbance measurements (measures absorbance at 260 nm and 260 nm/280 nm ratio)|
|Selectivity for DNA or RNA (Figure 3)||Accurately measure both DNA and RNA in the same sample||Results for samples containing both DNA and RNA are nondiscriminatory—you cannot distinguish one from the other|
|Accuracy and precision at low concentrations (Figure 4)||Accurately quantifies DNA in samples with concentrations as low as 10 pg/μL||Not recommended for concentrations under 2 ng/μL; variation for sample concentrations <10 ng/μL is often high|
|Sensitivity and range (Figure 5)||The effective range covers a sample concentration range of 10 pg/μL to 1 μg/μL DNA||Covers a sample concentration range of 2 ng/μL to 15 μg/μL; uses 0.5–2 μL of sample|
|Can indicate contamination||No||Gives peaks revealing the presence of contaminants|
A great study by Kapp et al., on “Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial” highlighted the variability between the two methods (table below)
“Nanodrop readings were 5.1-fold higher than Qubit measurements for the same samples”
Okay, so why does it matter?
Following extraction, accurate DNA yield must be determined in order to input the appropriate amount of material for downstream sequencing library preparation, as well as to understand theoretical allele frequency thresholds (see below). While spectroscopic methods like UV/VIS (eg. Nanodrop spectrophotometer) provide adequate estimation at concentrations higher than 10 ng/µL, fluorometric quantification (eg. qubit fluorometer) will always be more accurate and specific (measuring DNA only).
Qubit vs Nanodrop
Importantly, the method of quantification will greatly affect your results. As shown in the figure, across measurements collected by multiple laboratories, there is notable variability between DNA quantified spectrophotometrically by Nanodrop vs. fluorometrically by Qubit. While spectroscopic methods (UV-VIS) provide adequate estimation at concentrations higher than 10 ng/µl, fluorometric quantification will arguably be more specific (measuring only DNA via intercalating dyes), and more accurate for quantification.
That being said, spectroscopic analysis has other utility in evaluating DNA quality. Spectroscopic analysis to determine the 260/280 and 260/230 ratios can provide insight into potential contaminates in the sample, which may affect downstream library preparation. Similarly, the level of fragmentation, often defined by the DNA integrity number or DIN may also affect downstream sample processing.
The importance of accuracy (regardless of the method) is highlighted in my previous blog post on ‘the importance of personalized medicine’.