When It’s All About Quality: The Importance of Purity and Integrity Estimation
August 1, 2022 | By Dr. Laura Torres Benito
DNA and RNA extraction is the starting point of many processes in molecular biology, diagnostics, and genomics research. After isolation, the quantification and analysis of quality are necessary to ascertain your nucleic acid concentration and the suitability of your sample for further analysis. This quality control is essential for many applications such as NGS workflows, which require high-quality DNA, or transcriptomic analysis, which requires RNA at the best conditions. Frequently, it is more important to have good quality nucleic acid samples, with low fragmentation or degradation rate, than high quantities of them. In this article, we clarify what is relevant when we talk about nucleic acid quality and how to determine the purity and integrity of your samples.
What does “quality” mean? Be clear in your mind about your needs
Determining purity of nucleic acid samples
Additional information and tips for your purity assessments:
3) The concentration of the sample is rather important when measuring purity. Below 20 ng/µL the measurement is not reliable, and from 20 to 50 ng/µL it can still have a huge variability, which means that the most accurate calculation of absorbance ratio is starting at 50 ng/µL. At low concentrations, replicate measurements are recommended.
4) Nucleic acid measurements in water are inaccurate and highly variable. RNA ratios are 0.3 – 0.4 units lower than buffered and mildly alkaline samples. A good alternative for spectrophotometric analysis is using Tris buffer.
|Impurities||A260/A280 ratio||A260/A230 ratio||DNA concentration||Comments|
|Proteins||Slightly reduced||Strongly reduced||Weakly affected||Effect of protein contamination on purity ratio depends on NA concentration|
|EDTA||Unchanged||Increased||Unchanged||Its effect on the ratio decreases with high DNA concentrations|
|Slightly reduced||Slightly reduced||Strongly affected||Shift of 260 nm peak towards 270 nm at high concentrations|
||Slightly reduced||Strongly reduced||Strongly affected||Effect on A260/A230 ratio will be enhanced by the presence of GTC|
|Ethanol||Unchanged||Slightly reduced||Unchanged||Not clearly distinguished in absorbance spectra|
|Reduced||Strongly reduced||Strongly affected||Detected even at very low concentrations|
|Tween® 20||Unchanged||Strongly reduced||Weakly affected||Only detected at high concentrations|
|Slightly reduced||Reduced||Unchanged||Very little impact on downstream applications|
|GuHCl**||Mostly unchanged||Reduced||Unchanged||Only detected at high concentrations|
* GTC = guanidine thiocyanate; **GuHCl = guanidine hydrochloride