When It’s All About Quality: The Importance of Purity and Integrity Estimation

July 31, 2022 Dr. Laura Torres Benito
RNA, DNA

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

Nucleic acid preparation protocols must accomplish the product quality needed for the specific downstream analysis. “Quality”, though, can have many facets, so we recommend being clear in what your requirements are. Is it yield, integrity or purity that you need? DNA/RNA yield refers to the total amount of nucleic acid we obtain after the isolation. It calculates by multiplying the DNA concentration by the final total purified sample volume. When talking about integrity, one refers to DNA and RNA being mostly intact, with a low degree of fragmentation or degradation. Purity indicates the absence of additional components in the final throughput. Those terms are not interrelated, as a high yield does not imply adequate integrity and purity, and vice versa. However, depending on the method for measuring DNA , the purity of the sample plays a critical role in the final concentration result.

Determining purity of nucleic acid samples

Once nucleic acids are extracted and occasionally cleaned up, purity readings will provide information about the quality of the sample and its suitability for posterior applications. Spectrometry is the widely used technique to evaluate nucleic acid purity, as nucleic acids absorb ultraviolet light at 260 nm. Numerous substances, which are utilized during DNA/RNA extraction protocols, could influence the UV absorbance of the purified samples if not completely removed during the preparation. Non-ionic detergents (Triton™ X-100 and Tween® 20) are frequently used in binding buffers. Chaotropic salts (guanidine thiocyanate, GTC, and guanidine hydrochloride, GuHCl) are common components of the binding buffers. ETDA, frequently used in DNA elution buffers, could affect absorbance spectra. Additionally, phenol, TRIzol™ or other reagents used in RNA purification protocols can influence the absorbance readings. To a certain extent, ethanol, used in wash buffers, may also influence absorbance (Figure 1 and Table 1).

The most used indicator of sample purity are the ratios of the absorbance at 260 nm vs 280 nm (A260/A280) and 260 nm vs 230 nm (A260/A230). These two absorbance ratios might reveal whether contaminants and impurities are present in the sample and weather the extract is suitable for downstream applications. The A260/A280 ratio provides insight regarding the type of nucleic acid present (dsDNA or RNA) as well as a rough indication of purity. Values of 1.85 – 1.88 indicate pure dsDNA; and a ratio of around 2.1 is generally accepted as pure RNA. Contamination with protein, phenol or other reagent associated with the extraction protocol will give a reduced A260/A280 ratio (Figure 1 and Table 1).

Expected A260/A230 values are commonly reported in the range of 2.3 – 2.4 for dsDNA and 2.1 – 2.3 for RNA. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants that absorb at 230 nm. These include chaotropic salts such as GTC and GuHCl, EDTA, non-ionic detergents, proteins, and phenol (Table 1).

Other components such as various detergents (SDS), dyes (Indigo, gel loading), primers, dNTPs, or precipitates, could also be present in nucleic acid preparations, but rather than changing the absorbance, they could inhibit some following applications.

Absorbance spectra of common contaminants with DNA. Constant amount of DNA was used for the absorbance measurement (DNA concentration = 124.33 ng/µL). Samples were mixed with different components to visualize changes in the spectra profile and in the purity ratios. Absorbance ratios for pure DNA: A260/A280 = 1.9; A260/A230 = 1.95.

Additional information and tips for your purity assessments:

1) RNA will typically have a higher 260/280 ratio due to the higher ratio of uracil compared to that of thymine.

2)    Usually, it is necessary to examine not only the ratio values but also the absorbance spectra. Some components hardly modify the proportion between 230 or 280, and 260 nm, but exhibit a clear shift of the curve. For example, when phenol is present in high concentrations, a clear deviation towards 270 nm might be observed with a slight reduction of both purity ratios.
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.

Table 1. Effect of common impurities of nucleic acid samples in purity ratio.

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	Decreased	Unchanged	Its effect on the ratio decreases with high DNA concentrations
Organic solvents Phenol	Slightly reduced	Slightly reduced	Strongly affected	Shift of 260 nm peak towards 270 nm at high concentrations
TRIzol™/Chloroforms	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
Detergents Triton™ X-100	Reduced	Strongly reduced	Strongly affected	Detected even at very low concentrations
Tween® 20	Unchanged	Strongly reduced	Weakly affected	Only detected at high concentrations
Salts GTC*	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

Assessing the integrity of the genetic material

The results of numerous molecular screening and assay methods often rely on the overall integrity of the nucleic acid input material. High-integrity DNA helps ensure robust results in applications such as NGS, gene editing, cloning, but also prenatal diagnostic, in vitro fertilization, forensic science, plant and animal breeding, among others. Damage and fragmentation of the DNA might lead to inaccurate results on your downstream applications. RNA integrity also refers to the conservation of the molecules after the extraction process. Total RNA extracts usually contain rRNA subunits, mRNA, tRNA, and small RNAs. RNA is inherently susceptible to RNase degradation, and it is a chemically unstable molecule. If you are working with RNA, quality is essential for RNA-based analysis like microarray technology and real-time RT-qPCR. Therefore, it is recommended to assess integrity in any case.

For decades the only way to determine nucleic acid fragmentation and degradation was the use of agarose gel-based electrophoresis, but this method is variable, inaccurate, and time-consuming. To assess DNA fragmentation level, quality ratio (Q ratio) can be calculated. Q ratio is determined using qPCR to amplify fragments of 3 different lengths: short (41 bp), medium (129 bp), and large (305 bp). The ratios of these fragments determine the total fragmentation rate and refer to the proportion of 129 bp or 305 bp fractions compared to the 41 bp fragments (Q129/Q41 and Q305/Q41, respectively). The Q ratio should range between 0 and 1. When DNA is degraded and cross-linked, so that it does not amplify properly during the PCR reaction, the number of larger amplicons would be lower, yielding a lower Q ratio. If the DNA does not have damage that interferes with PCR, there will be equivalent amplification of both, the larger and the shorter amplicons, bringing the Q ratio closer to 1.

Automated platforms that determine nucleic acid quality have been developed to save time, costs, and materials. Currently, those systems consist of an automated and miniaturized electrophoresis system, realized by lab-on-chip technology. It includes a microfluidic system, combined with computer-controlled instruments and measurements and a software-based report and analysis. Those platforms determine DNA integrity via DIN (DNA Integrity Number) and RNA integrity via RIN (RNA Integrity Number). The DIN and the RIN are based on a numbering system from 1 to 10, where 1 represents the most degraded nucleic acid profile, and 10 is the most intact one.

If you aim to achieve a high-quality nucleic acid final product, BioEcho Life Sciences has developed an innovative technology that allows DNA and RNA extraction in a single step, avoiding carryover contaminants, cellular debris, and inhibitors of downstream applications. During the isolation process, nucleic acids freely flow through the matrix, which traps any other components. The procedure provides active enzymes that improve lysis efficiency eliminating mechanical disruption and overnight lysis. Moreover, it simplifies the workflow with a subsequent single purification step under aqueous conditions, avoiding the use of additional components and the risk of nucleic acid damage from repeated centrifugation. We ensure reduced handling during extraction protocols which leads to a decreased nucleic acid degeneration, contaminants, and inhibitors. At the end, a highly pure and intact genetic material is obtained for successive applications. Learn more about our nucleic acid extraction technology.

Author: Dr. Laura Torres Benito

Laura is a passionate scientific communicator, with an extensive experience as a researcher in several fields, including human genetics, biotechnology, and neuroscience. Since joining BioEcho in 2022, she enjoys creating appealing content and material for our customers and interested parties. Laura likes practicing yoga, cooking wholesome Mediterranean food, and playing the piano. She also loves spending weekends on the nature, rock climbing and hiking.