Nucleic Acid Quantification: Methods and Considerations

Nucleic acid quantification is a foundational technique in molecular biology research. Jennifer Mook, Senior Applications Scientist at Promega, notes that “nucleic acid quantification is important in determining if the nucleic acid extracts are acceptable for use in downstream applications. Importantly, the success of molecular cloning, next-generation sequencing, and nucleic acid amplification assays depends on knowing nucleic acid concentrations.”

These experiments also have societal impacts. Many key applications use gene quantification to “detect and diagnose microbial and viral diseases, to identify cancer types, or develop pharmaceutical treatments and cutting-edge synthetic biology products,” states Constantine Garagounis, Product Development & Marketing Specialist at PCR Biosystems. Andrew Jones, Market Development Manager at DeNovix, also adds that “forensics, pharmacogenomics, and environmental studies use nucleic acids in their workflows and require precise measurements.”

The importance of measuring nucleic acid concentrations has encouraged the development of molecular approaches to quantify them. Each of them has their applications and technical considerations that impact its applicability with different kinds of samples. Knowing these aspects will help scientists attain reproducible downstream molecular biology research.

Spectrophotometry

Spectrophotometers measure nucleic acid concentrations based on absorbance values. Absorbance refers to the extent to which a compound absorbs light. The linear correlation between absorbance and nucleic acid concentrations—thanks to the Beer-Lambert Law—makes nucleic acid quantification possible.¹ Jones explains this further, “Spectrophotometers measure absorbance values at 260 nm, the wavelength where nucleic acids absorb the most light. They then use the linear relationship between absorbance and concentration to calculate nucleic acid abundance. This approach provides several benefits. For one, spectrophotometers do not need reagents to measure nucleic acid levels. Moreover, sample volumes as small as 1 uL are also enough to perform spectrophotometer-based quantification.”

Several technical considerations when using the spectrophotometer must, nonetheless, be made. First, the linear relationship observed in the Beer-Lambert Law falls apart when the absorbance of a 1-cm path length solution exceeds ~1.5–2 absorbance units.² In such cases, the extracts would need to be diluted first before redoing the measurements. Jones adds that “spectrophotometers may also struggle to detect DNA in samples with low nucleic acid concentrations. Nevertheless, the DeNovix microvolume spectrophotometer can detect nucleic acid concentrations as low as 0.75 ng/uL.” Even so, quantifying nucleic acids in ultra-dilute samples would require other approaches for measuring nucleic acids.

Fluorometry

Fluorometry represents one appealing alternative for nucleic acid quantification. It uses dyes that intercalate with diverse species of nucleic acid fragments, including double- and single-stranded DNA and RNA.³ The dyes themselves have low intrinsic fluorescence, only lighting up when they bind to the biological molecules. Mook points out that “because unbound dyes do not fluoresce, they provide a low background and a broad dynamic range for measuring nucleic acid concentrations.” The specificity that fluorometry affords provides it with 10–1000 times more sensitivity in detecting nucleic acids compared with spectrophotometry.⁴ This feature makes fluorometry capable of detecting nucleic acids even in highly dilute samples.

Multiple variables must be considered to ensure the success of fluorometry-based quantification. Jones lists these variables in detail, “First, the fluorophore must cover the range of concentrations likely to be obtained from the samples being studied. Scientists should also consider whether the fluorometer’s excitation source, along with its excitation and emission filters, match the profiles of the fluorophores. Quantification kits should also be specific for the analyte of interest, without adverse effects from fragmented nucleic acids and contaminants. Finally, appropriate standards must be prepared for the samples as fluorophores can bind differently to DNA depending on GC content.”

qPCR-based quantification

The technologies mentioned were developed to determine total nucleic acid concentrations. However, a growing body of research raises the need to measure the abundance of specific targets. Quantitative PCR (qPCR) is a foundational technique for quantifying these nucleic acids. It can measure DNA biomarkers, compare expression patterns for specific genes, and serve as quality controls for cloning experiments.⁵ qPCR accomplishes this either by quantifying absolute genome copy numbers⁶ or measuring relative changes in gene expression.⁷

Several controls are necessary for qPCR experiments to succeed. Negative, no-template controls are “necessary for any valid quantification method. They assure that amplification does not take place due to contamination,” Garagounis poignantly points out. He also adds other controls that should be used: “In RNA experiments, no-RT (reverse transcription) controls can help identify amplification due to contaminating DNA because the controls were not exposed to reverse transcription before qPCR thermocycling.”

Nucleic acid purity and integrity

Even as methods for nucleic acid quantification exist, scientists also need ways to determine the intactness of nucleic acid extracts. Mook details one instance where nucleic acid degradation plays a role in downstream experiments: “DNA- and RNA-sequencing require input nucleic acids to be a specific length. The chemical and fixation processes underlying archived formalin-fixed paraffin-embedded (FFPE) samples result in severely degraded nucleic acids after purification. Many of the nucleic acid fragments can become too short for sequencing. This adversely affects the quality of sequencing data.” Additionally, as the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines note, adverse effects can arise when using degraded RNA to quantify lowly abundant transcripts.⁹ Mook elucidates on this further, adding that “PCR assays amplifying longer fragments may prove unsuccessful when using degraded nucleic acids as templates.”

For both cases, multiple technologies designed to measure nucleic acid integrity exist. The Bioanalyzer and TapeStation systems are the most used technologies. Both of them rely on capillary electrophoresis to separate charged molecules by size.⁹ From the electrophoresis, a plot of fluorescence intensity versus migration time is generated. Subsequent software tools can then assess nucleic acid integrity based on the shapes of the plots. For instance, RNA integrity is measured with the RNA integrity number, where 10 is fully intact and 1 is fully degraded.¹⁰ These metrics provide researchers with the ability to identify degraded nucleic acids and ensure that fragments are of ideal length for downstream experiments.

Conclusion

Measuring the concentrations of nucleic acids in extracts is an important component of a successful molecular genetics study. Accurately measuring nucleic acid concentrations requires a suite of lab tools used together. Some of these tools measure total nucleic acids. Fluorometry provides more sensitivity and specificity in nucleic acid quantification, while spectrophotometry can assess extract contamination. Other tools, namely qPCR, measure total copy numbers for specific genes. Although both have their uses, each must be accompanied by quality control measures to ensure nucleic acid purity and integrity. Accounting for all these variables will ensure the success of any molecular genetics study.

References

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