Quantifying DNA and RNA accurately is a prerequisite for reproducible molecular biology. Two main approaches dominate: spectrophotometry and fluorometry.
UV Spectrophotometry (NanoDrop)
The NanoDrop spectrophotometer measures 1–2 µL of sample placed directly on a pedestal, using surface tension to create a defined path length (0.2–1.0 mm). It measures absorbance at 230, 260, and 280 nm:
- A₂₆₀ measures nucleic acid concentration. For dsDNA, 1 OD₂₆₀ = 50 ng/µL; for RNA, 1 OD₂₆₀ = 40 ng/µL; for ssDNA, 1 OD₂₆₀ = 33 ng/µL.
- A₂₈₀ measures protein and phenol contamination. A pure DNA sample has an A₂₆₀/A₂₈₀ ratio of ~1.8; pure RNA ~2.0.
- A₂₃₀ measures organic compounds, chaotropic salts, and phenol. The A₂₆₀/A₂₃₀ ratio should be ~2.0–2.2 for pure nucleic acids.
The NanoDrop is fast and requires minimal sample but cannot distinguish DNA from RNA and is affected by nucleotides, single-stranded fragments, and other UV-absorbing contaminants.
Fluorometric Quantification (Qubit)
The Qubit fluorometer uses fluorescent dyes that bind specifically to dsDNA, RNA, or protein. When bound, the dye fluoresces strongly, and the fluorescence intensity is proportional to the concentration.
Qubit assays are highly selective — the dsDNA assay does not detect ssDNA or RNA, and the RNA assay does not detect DNA. They are also more sensitive than spectrophotometry, detecting concentrations as low as 0.1 ng/µL. The trade-off is cost per assay (reagents are dye-based) and the need for two calibration standards per assay.
Choosing a Method
Use spectrophotometry for routine quantification of purified plasmid DNA, PCR products with no protein contamination, and checking purity ratios. Use fluorometry for precious samples (NGS library prep, ChIP DNA), when the sample contains nucleotides or degraded fragments, or when you need to specifically quantify dsDNA in the presence of RNA.
Agarose Gel Quantification
For semi-quantitative assessment, run the sample on an agarose gel alongside a ladder with known band intensities (e.g., 100 ng, 50 ng, 25 ng). Ethidium bromide or SYBR Safe fluorescence intensity correlates roughly with mass. Digital gel imaging systems can integrate band intensity for an estimate, but this method is less accurate than spectrophotometry or fluorometry.
