How to Evaluate Nucleic Acid Quality After Extraction

Nucleic acid extraction is only just the first step in a typical molecular workflow. Assessing the quality of the purified DNA or RNA material is a necessary follow-up step as this has direct consequences for sensitive downstream applications, such as PCR-based assays and sequencing. In this article, we review how assessing concentration, purity, and integrity, alone and in combination, is important for generating reliable results, along with some expert recommendations. 

Quantifying the sample

For post-extraction quantification, common methods include microvolume UV spectrophotometry, fluorometry with dye-binding assays, PCR, and electrophoresis. Spectrophotometry devices like the NanoDrop and DeNovix are fast, require no specialized reagents, and can report purity ratios alongside concentration.^1,2  However, UV absorbance cannot readily distinguish intact double-stranded DNA from RNA, single-stranded DNA, or free nucleotides, which can lead to concentration overestimation up to 3- or 4-fold higher than fluorometric methods in raw extracts.^1,2  At low concentrations, fluorometry is more specific and sensitive, although it cannot detect non-nucleic acid contaminants such as protein, phenol, or carbohydrate carryover.

With both spectrophotometry and fluorometry, the readings are generally reliable even after freeze-thaw cycles and is generally consistent across platforms regardless of the operator.^1,2  However, the type of sample can influence the impact of these readings. For example, fragmented material from samples like formalin-fixed, paraffin-embedded (FFPE) tissue may appear to be sufficient in terms of concentration, yet still perform poorly in enzymatic steps.^3,5  Low-input workflows and PCR-dependent assays further benefit from fluorometric or PCR-based quantification, which heavily relies on amplifiable or double-stranded DNA templates.^1,2,3

Measuring purity

Purity assessment after extraction often relies on UV absorbance ratios, most commonly A260/280 and A260/230. A260/280 values below ~1.7–2.0 generally indicate protein or phenolic contamination and A260/230 values below ~2.0–2.2 can signal residual salts or organic solvents.^1,2,5  These ratios show strong correlation and minimal variation between different microvolume spectrophotometric platforms like DeNovix and NanoDrop.¹  Measuring purity is very important, as residual guanidine, phenol, and reagents can inflate the sample’s apparent concentration, making them appear to have high yield, but still causing issues in downstream enzymatic and sequencing procedures.^1,2  

Users should also be aware that absorbance values are sensitive to measurement conditions and can shift with a mismatched blank, low sample concentration, or saline elution buffers. Sample origin can also lead to unique spectral behaviors in certain cases. For example, DNA extracts from fish tissue often contain non-DNA compounds that absorb UV light. These contaminants can artificially inflate yield estimates spectrally, even when purity ratios appear completely normal.²  Inversely, the use of specialized eluents can skew ratios, making them appear impure but without actually negatively impacting downstream sequencing.⁵  

Assessing integrity

Nucleic acid integrity refers to sample templates maintaining sufficient fragment lengths as well as the chemical and functional properties necessary for downstream enzymatic assays and sequencing.^3,4,5,6  Integrity scoring is commonly performed by automated electrophoresis devices using algorithms that convert fragment size distributions into a numerical score, such as an RNA Integrity Number (RIN) or DNA Integrity Number (DIN).^3,4  Another metric is DV₂₀₀, which measures the percentage of RNA fragments longer than 200 nucleotides, ideal for evaluating the broader size distribution of all RNA fragments in a sample. ⁵  

Automated integrity scores summarize fragment size distributions into a single metric, allowing objective sample comparisons for predicting downstream assay performance. A low RIN score for instance, can indicate partially degraded samples that can lead to reduced library complexity, shorter read lengths, decreased full-length read coverage, and an overrepresentation of shorter genes and isoforms in RNA sequencing.⁴  Another example is in panel sequencing of clinical FFPE tumor samples, where a DIN cutoff of 2.5 has been found to separate successful from failed NGS analyses.³

Functional DNA integrity can also be assessed via qPCR-based amplifiability assays, which measure amplifiable template rather than relying solely on physical size. Because degraded DNA impairs longer amplicons more severely than shorter ones, quantifying the amplification efficiency ratio of the longer target over the shorter target can be used to confirm that the sample is both sufficiently intact and free of enzymatic inhibitors. ³

Complementary methods

For a more comprehensive assessment of sample quantity, purity, and integrity, a more reliable approach would be to use multiple methods in combination. Nish Kumar, Global Product Marketing at Bio-Rad Laboratories, describes how this layered strategy is applied within the company.

“Within the R&D teams, our QA/QC approach combines three complementary measurements. UV spectrophotometry gives a quick concentration estimate and purity ratios (A260/280 for protein, A260/230 for salts and organics). Automated electrophoresis with instruments such as an Agilent Bioanalyzer or agarose gel imaging confirms integrity, using an RNA integrity score for RNA and a DNA integrity score or high-molecular-weight band profile for DNA. Finally, functional assays such as qPCR or Droplet Digital PCR confirm that samples amplify efficiently and are free of inhibitors. These methods were chosen because no single metric captures quantity, purity, and integrity, and in the end, a functional assay is the best predictor of downstream success.”

In the end, according to Kumar, “a functional assay is the best predictor of downstream success.” Such tests are particularly ideal as a substitute or complement when a standard quality check does not suffice. For example, Kumar recommends the following for RNA samples: “The main suggestion in post-extraction of the sample is to protect RNA integrity by stopping RNase activity at the moment of collection and then add a quick integrity check using an electrophoresis assay wherever the sample’s condition can change. Bio-Rad’s PrimePCR controls are an example of a functional check for genomic DNA contamination that works even when a sample can't be run on electrophoresis.”

High quality extractions

Beyond measuring quality after extraction, some approaches are designed to prevent specific quality problems from arising in the first place. This can include specialized kits incorporating methods and components designed to combat the root causes of quality loss.

“For gene expression applications using cultured cells, the SingleShot Cell Lysis Kit takes the unique approach of skipping RNA isolation altogether. Cells are lysed directly in a PCR plate, and the lysate goes straight into RT-qPCR in about 20–30 minutes,” recommends Kumar.  “DNase removes genomic DNA, which addresses purity. Proteinase K digests proteins that can inhibit reverse transcription and PCR, another purity problem. An RNase inhibitor protects RNA during lysis, which addresses integrity. Because there is no column step, the kit also solves a concentration problem: rare transcripts and RNA from very small samples aren't lost to binding and elution.”  

Ertan Ozyamak, Ph.D., Global Product Marketing at Bio-Rad Laboratories, recommends Chelex 100 Molecular Biology Grade Resin and the related InstaGene Matrix as an alternative to the bind-wash-elute chemistry of typical column kits. These reagents protect nucleic acids by limiting nuclease activity, offering a fast, simple, and cost-effective way to prepare nucleic acids in a single tube, with fewer handling steps. The resulting samples are suitable for downstream detection by PCR, RT-PCR, ddPCR, or LAMP.

“Chelex 100 is a polymer that strongly binds divalent metal ions. By removing cations such as Mg²⁺ that metallo-nucleases require for their activity, it protects DNA and RNA integrity. InstaGene Matrix applies the same chemistry as a ready-to-use Chelex suspension,” explains Ozyamak.

Advice for addressing quality issues

Kumar offers useful recommendations for addressing common quality issues after sample extraction:

Low yield. Common causes are too little starting material, incomplete disruption, or too much material that overloads the column. Stay within the recommended input range and try to improve tissue disruption if needed. Confirm ethanol was added to wash buffers. For low eluate volume, an additional 1–3 minutes of elution spin time can help.  If you still can’t avoid low yield, then consider adding a pre-amplification step before performing your qPCR/ddPCR reaction.

Low A260/230 ratios. Usually caused by guanidine or phenol carryover. An extra wash and dry spin before elution can help. Guanidine contamination may not affect downstream performance, so use a functional assay to determine whether cleanup is necessary.

Concentration discrepancies. UV can overestimate concentration because many contaminants, free nucleotides, or degraded fragments absorb at 260 nm. When UV and fluorescence disagree, fluorometric measurements are generally more reliable. For critical applications, use fluorescence or ddPCR.

Degraded RNA. Degradation can result from environmental RNases or RNases released before lysis. Use RNase-free materials, decontaminate surfaces, and work quickly. The PrimePCR RNA Quality Assay detects degradation by comparing amplification of two products from the same transcript.

Genomic DNA contamination. This may appear as amplification in no-RT controls or unexpectedly high expression. Causes include incomplete or inactive DNase treatment and overloaded columns. Extend DNase incubation, avoid repeated freeze/thaw, and reduce sample input. The PrimePCR DNA Contamination Control Assay can compare gDNA levels across samples.

Downstream inhibition. A sample can pass purity checks but still inhibit qPCR. A dilution test can reveal inhibition: smaller-than-expected Cq shifts after dilution suggest inhibitors. PrimePCR Positive PCR and Reverse Transcription Controls can help identify whether the problem is the sample, RT, or PCR.

As a general advice for these issues, always match the diagnostic to the symptom you’re trying to detect. For example, run UV ratios for contamination, evaluate fluorescence or ddPCR for concentration, and include functional qPCR controls for integrity and inhibition. Then trace the issue back to the most likely problematic step.

Final takeaway

The quantity, purity, and integrity of extracted nucleic acids ultimately depend on the sample type, experimental conditions, and operator technique, so each preparation can have its own unique properties. For the best results in the intended application, whether an assay or sequencing, users should assess sample quality with methods suited to each attribute, while also taking into account any sample-specific nuances. 

References

1. Versmessen N, Van Simaey L, Negash AA, et al. Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts. PLoS One. 2024;19(6):e0305650. doi:10.1371/journal.pone.0305650

2. Bruijns B, Hoekema T, Oomens L, Tiggelaar R, Gardeniers H. Performance of Spectrophotometric and Fluorometric DNA Quantification Methods. Analytica. 2022; 3(3):371-384. https://doi.org/10.3390/analytica3030025

3. Hiramatsu K, Matsuda C, Masago K, et al. Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling. Am J Clin Pathol. 2023;160(3):261-267. doi:10.1093/ajcp/aqad046

4. Prawer YDJ, Gleeson J, De Paoli-Iseppi R, Clark MB. Pervasive effects of RNA degradation on Nanopore direct RNA sequencing. NAR Genom Bioinform. 2023;5(2):lqad060. doi:10.1093/nargab/lqad060

5. Decruyenaere P, Verniers K, Poma-Soto F, Van Dorpe J, Offner F, Vandesompele J. RNA Extraction Method Impacts Quality Metrics and Sequencing Results in Formalin-Fixed, Paraffin-Embedded Tissue Samples. Lab Invest. 2023;103(2):100027. doi:10.1016/j.labinv.2022.100027

6. Dahn HA, Mountcastle J, Balacco J, et al. Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing. Gigascience. 2022;11:giac068. doi:10.1093/gigascience/giac068