Blog article
Considerations related to purity and quality control of isolated nucleic acids
Written by Tonje Steigedal, PhD, CEO of Lybe Scientific
4. september 2025
Absorbance measurements and especially the A260/280 and A260/230 ratios are commonly used to assess the purity of nucleic acid samples.
The A260/280 Ratio has ideal values at ~1.8 (DNA) and ~2.0 (RNA) and alterations from this typically indicate protein contamination. However, the value cannot distinguish between different types of protein and between different types of protein contamination. Furthermore, the number can also be sensitive to pH and ionic strength of the solution.
The A260/230 Ratio should ideally be between 2.0 – 2.2 for pure nucleic acids and alterations from this are normally an indication of contamination from organic compounds and salts (e.g., phenol, guanidine, EDTA). The A260/230 ratio can be highly variable depending on extraction method and buffer composition.
Another important issue to consider is that low values may not necessarily correlate with poor downstream performance. We did a test to illustrate this point in figure 1 below.
Figure 1: Correlation between A260/280 and A260/230 ratios and DNA concentration. A) Concentration of DNA [ng/µl] in samples with increasing sample input, B) DNA concentration [ng/µl] in serial dilution of substrate, C) A260/280 and A260/230 ratios of samples with increasing sample input and D) A260/280 and A260/230 ratios in serial dilution series of eluates.
The figure clearly shows that the A260/280 and A260/230 ratios typically move out of the ideal range once the concentration drops.
Corresponding qPCR data shows that off-range A260/280 and A260/230 ratios does not correspond with poor qPCR results.
Figure 2: Showing housekeeping gene Ct-values from qRT-PCR run on the extracted nucleic acids from figure 1A, increasing sample input. Values correlate very well with expected values based on dilution factor.
We strongly encourage to use complementary Methods for additional quality control depending on application. Fluorometric quantification (e.g., Qubit) can be used for more accurate concentration and gel electrophoresis or Bioanalyzer may be used for more detailed information about nucleic acid integrity.
Most importantly, we encourage users to run either qPCR, RT-qPCR or other downstream applications for functional validation as absorbance ratios don’t necessarily show the whole picture.
