Across all areas of life science research, scientists are increasingly aware of the need for the absolute quantification of nucleic acids. To make their analyses actionable, they want to directly measure copies of a molecule, rather than rely on a simple positive or negative result. The challenge, however, is that any tool that quantifies nucleic acids needs to be standardized against a universal reference material produced by an authoritative body such as the World Health Organization. If this practice is not in place, the results obtained using this tool will not be reproducible across different tests, laboratories or locations.
Currently, there are no convenient primary reference measurement procedures for quantifying these standards. As a result, it is difficult for research laboratories and diagnostics manufacturers to harmonize their nucleic acid quantification methods and produce reliable assays. Fortunately, several metrology laboratories – labs that study the science of measurement – are...
Reliable RNA and DNA Quantification
Developing a universal reference measurement procedure would impact every lab that handles nucleic acids. However, one area where absolute RNA and DNA quantification would be especially useful is in clinical laboratories. To create a reliable in vitro diagnostic, for instance, it needs to be calibrated against a standard that can be traced back to an SI unit. This allows researchers to compare the resulting data directly against different labs to ensure accurate interpretation.
Creating a traceable diagnostic starts with a hierarchy of successive calibration steps that ensure each test reports a true quantity based on a certified reference material (CRM) from a national metrology lab. At the top of the hierarchy, metrologists calibrate a primary reference measurement procedure against the CRM. This primary procedure ultimately supports the calibration of a diagnostic test and controls. Each step in this hierarchy introduces uncertainty, so, according to the Consultative Committee for the Amount of Substance (CCQM) of the International Bureau of Weights and Measures (BIPM), the primary reference measurement procedure must have “the highest metrological properties.”1 It must operate according to a completed, described, and understood protocol, and must have its uncertainty completely written down in terms of SI units.
“Any method that's used as part of a primary reference measurement procedure, at its most accurate, would be SI-traceable,” said Jim Huggett, Ph.D., Principal Scientist (Nucleic Acid Research) in LGC's Health Science and Innovation Division and Senior Lecturer in Analytical Microbiology at the University of Surrey. Put another way: Its calibration should trace back to a universal standard, or CRM.
An SI-traceable reference measurement system for nucleic acids would benefit virtually all areas of research and medicine that rely on nucleic acid quantification. In medicine, perhaps the biggest long-term impact will be seen with DNA quantification in cancer. Accurately quantifying tumor load via circulating tumor DNA allows physicians to track both disease progression and treatment response.
But for now, the focus of the world and the greatest need for absolute quantification concerns the COVID-19 pandemic. As authorities try to mitigate the spread of the virus, accurate quantification of SARS-CoV-2 viral loads in patients is critical. This information helps authorities track the course of the disease and determine whether someone is contagious, with or without clinical symptoms. In the absence of a reference standard, qPCR is only able to produce a qualitative result (i.e. Ct values). Digital PCR, on the other hand, can fully quantify the virus in copies/μl.2
Variable qPCR Standards
In all of these areas, the current go-to method for quantifying primary reference materials is real-time quantitative polymerase chain reaction (qPCR). However, qPCR does not offer absolute quantitation. To interpret a qPCR result in terms of nucleic acid quantity, users need to generate a standard curve using a serial dilution, a procedure that is prone to error and bias.
“RT-PCR has played a big role in helping us quantify molecular DNA and RNA, and it’s used extensively,” says Dr. Huggett. “But in reality, it’s actually quite difficult to get a precise measurement and to know the trueness of the result because it’s on a log scale. If you count 500 molecules, is it truly 500 molecules, or is it 50 or 5000? That’s the type of variation you could see.”
In fact, digital PCR has already been used to quantify qPCR reference standards, exposing qPCR’s variability and inconsistency. This variability makes qPCR less sensitive to rare genetic variants associated with cancer. It also performs poorly when quantifying minimal residual disease, as seen with low ctDNA concentrations or viral load, further complicating its role as a reliable and reproducible quantification method.
Another source of qPCR variability stems from differing amplification efficiencies of individual assays. Researchers must interpret the quantity of a target nucleic acid sequence in a sample based on sequence amplification and the subsequently-generated fluorescent levels. This can be an unreliable process, with several factors that can interfere with the results. For example, samples containing highly variable sequences or mismatches between primers or probe sequences, as well as secondary and tertiary nucleic acid structures, can hamper the quantification of the target sequence. qPCR can also be impacted by inhibitors in the sample that reduce the overall level of amplification and affect the final quantification. For qPCR to be accurate, it must be calibrated using a standard curve, such as those produced using a primary reference measurement protocol that involves digital PCR.
Digital PCR as a Primary Reference Measurement Tool
Unlike qPCR, digital PCR doesn’t require calibration; rather, it directly quantifies nucleic acid samples, making it eligible as an SI-traceable primary reference measurement procedure. Digital PCR works by directly counting the number of nucleic acid molecules in a sample, bypassing the need for a standard curve, and making it more precise and reliable. The method uses a droplet reader to digitally count the number of target sequence copies in a sample that has been partitioned into tens of thousands of nanoliter-sized droplets. “Count” is a recognized dimensionless SI unit, which means digital PCR could potentially serve as an SI-traceable primary reference measurement procedure for counting DNA copy number concentration.
Over the past several years, metrology labs across the globe have demonstrated the accuracy and sensitivity of digital PCR in many areas of research, supporting its potential role as a primary reference measurement procedure. For example, David Dobnik, Ph.D. and his team at the National Institute of Biology, in Ljubljana, Slovenia have even shown that dPCR can accurately and precisely quantify viral titers in plants and genetic modifications in foods.3,4
Along with quantifying primary reference standards, digital PCR is being explored as a tool for evaluating the accuracy and clinical utility of laboratory developed tests (LDTs). The National External Quality Assessment Service (NEQAS) in the UK, for example, uses digital PCR to score LDTs from 1,500 testing labs around the world. Sandi Deans, Ph.D., a member of the UK NEQAS Consortium, believes the biggest impact digital PCR will have as a reference measurement tool in medical testing labs is in cancer.
“We do a lot of tumor testing, and tumor DNA tends to be present at low levels,” Deans explained. “We need to make sure we’ve got as much good-quality DNA present as possible to get a reportable result.” Digital PCR helps Deans and her colleagues at NEQAS determine if the samples being used for medical testing contain sufficient quality DNA to produce a true and actionable result. “Detecting these ctDNA molecules means the difference between identifying sequence variants in tumors that can be treated using a personalized approach, or not,” said Deans.
What Accurate DNA Quantification Means for the Future of Medicine
At its core, a reliable primary reference measurement procedure is the key to many forms of measurement across a diverse range of scientific research. In measuring nucleic acids, accurate calibration ensures that life science researchers and physicians can achieve a result they can trust, publish, and use to make clinical decisions that change lives. With its ability to quantify nucleic acids without the need for calibration, digital PCR fits this need and will likely become an essential tool for laboratories around the world to calibrate their measurements against. We cannot yet predict the full impact of digital PCR's adoption as a primary reference measurement tool or understand the extent to which the traceability of nucleic acid measurements will affect life science research, but it will undoubtedly bring positive change to clinical molecular laboratories and hospitals around the world.
Read more about the process of establishing digital PCR as an SI-traceable primary reference measurement tool in the whitepaper "Counting DNA Molecule by Molecule".
- Bunk DM. Reference materials and reference measurement procedures: an overview from a national metrology institute. Clin Biochem Rev. 2007;28(4):131-7.
- Liu X, Feng J, Zhang Q, et al. Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets. Emerg Microbes Infect. 2020;9(1):1175-1179.
- Mehle N, Dobnik D, Ravnikar M, Pompe novak M. Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains. Anal Bioanal Chem. 2018;410(16):3815-3825.
- Dobnik D, Spilsberg B, Bogožalec košir A, Holst-jensen A, Žel J. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction. Anal Chem. 2015;87(16):8218-26.