cfDNA extraction: Here’s what you need to know

The Takeaway: Cell-free DNA is an important tool that researchers can use to detect cancer. But exactly what is it and how do researchers obtain it? This blog explains more.

What is cfDNA?

Cell-free DNA (cfDNA) is a term that describes the different types of DNA that circulate in the bloodstream. These short DNA fragments (usually less than 200 base pairs) are released into the bloodstream when cells die or through active secretion. During pregnancy, cfDNA can be released from the mother’s tissue and placenta. Tumors can also shed cfDNA, in that case it is often called circulating-tumor DNA (ctDNA).

Extraction of cfDNA can be difficult to ensure isolation of the minor fraction of cell-free DNA compared to the more prevalent DNA from inside cells in the sample. The assessment of cell-free DNA (cfDNA) extraction involves evaluating the efficiency, purity, and yield of the extracted DNA. cfDNA is useful for screening tests that can estimate the risk of a fetus having conditions such as Down syndrome. It is also important for early cancer detection, cancer monitoring, and potential use for targeted therapy selection when a tissue sample isn’t available. Testing, however, is complex, and can be challenging for labs to ensure it is not producing false positives or false negatives.

What is cfDNA extraction?

Cell-free DNA extraction is a laboratory process that involves isolating and purifying DNA fragments that circulate freely in the bloodstream or other bodily fluids. cfDNA is derived from cells undergoing apoptosis (a process that causes a cell to die), necrosis (uncontrolled cell death that results in tissue damage), or actively releasing fragmented DNA into the extracellular environment. It is a valuable source of genetic material for various applications, including non-invasive prenatal testing (NIPT), and cancer diagnostics, monitoring, and therapy section.

The process of cfDNA extraction typically includes the following steps:

• Sample collection: Blood is commonly used for cfDNA extraction, although other bodily fluids such as saliva, urine, or cerebrospinal fluid can also be sources of cfDNA.
• Cellular debris removal: The collected sample is often initially processed to remove cellular components, such as red and white blood cells. This step may involve centrifugation or filtration.
• Plasma or serum separation: The remaining liquid component, which is usually plasma or serum, is separated from the cellular debris. Plasma is often preferred for cfDNA extraction because it contains a higher concentration of cfDNA.
• cfDNA extraction: Various methods can be used to extract cfDNA from the liquid component. Common techniques include silica membrane-based column purification, magnetic bead-based purification, and other commercially available kits.
• Purification: The extracted DNA can be further purified to remove contaminants, proteins, and other impurities. This step is crucial for obtaining high-quality cfDNA.
• Quantification: The concentration of cfDNA in the extracted sample is measured using methods such as fluorometry or spectrophotometry to provide information about the yield of cfDNA from the sample.
• Quality assessment: The quality of the extracted cfDNA is assessed by examining factors such as purity ratios (e.g., 260/280 and 260/230 ratios).
• Storage: The purified cfDNA can be stored for subsequent analysis. Proper storage conditions, such as low temperature and protection from degradation, are essential to maintain the integrity of cfDNA.

Applications of cfDNA extraction

cfDNA extraction is a critical step in various molecular biology applications, including the detection of genetic mutations, epigenetic changes, and chromosomal abnormalities. Advances in extraction techniques have improved the sensitivity and specificity of downstream analyses, making cfDNA extraction a key component in the field of non-invasive diagnostics.

Key factors to consider when assessing cfDNA extraction

There are some key factors to keep in mind when assessing cell-free DNA extraction methods to ensure efficient and reliable extraction. NGS is the most common use for cfDNA analysis due to its sensitivity, and having a high-quality library prep chemistry is important. These factors include:

Yield

The amount of cfDNA extracted is crucial as it determines the downstream applications' sensitivity and success. High yield extraction methods are desirable to maximize the amount of cfDNA obtained from a given sample.

Quantification: Measure the concentration of the extracted cfDNA using a fluorometer or spectrophotometer. This indicates the amount of cfDNA obtained per unit volume.

Yield consistency: Assess the consistency of cfDNA yield across different samples and extraction batches. Consistency is crucial for reproducibility in research applications.

Purity

The extracted cfDNA should be free from contaminants such as proteins, RNA, and genomic DNA. High purity ensures accurate downstream analysis, particularly in sensitive applications like next-generation sequencing (NGS) and PCR.

260/280 and 260/230 ratios: Use spectrophotometric measurements to determine the 260/280 and 260/230 absorbance ratios. A ratio close to 1.8 for 260/280 and 2.0 for 260/230 indicates good purity, suggesting minimal contamination by proteins, phenol, or other impurities.

Recovery efficiency: Comparison with gold standard: If available, compare the cfDNA extraction method with a gold standard or well-established method to evaluate the efficiency of the new protocol.

Inhibitor removal

PCR performance: Assess the performance of downstream applications, such as PCR, to ensure that inhibitors from the extraction process are not affecting the results.

Sample-to-sample consistency: Evaluate the uniformity of cfDNA extraction across different sample types (e.g., plasma, serum) and conditions (e.g., fresh/frozen samples).

Automation and throughput: Consider the ease with which the extraction process can be automated, especially for high-throughput applications.

Time and cost

Extraction time: Evaluate the time required for the extraction process. Some applications may require a quick turnaround. Rapid extraction methods are advantageous, especially when processing large numbers of samples or when timely results are needed. However, speed should not compromise yield, purity, or reproducibility.

Cost-effectiveness: Consideration of the overall cost per sample, including reagents, consumables, and equipment, is important, especially for high-throughput applications. Cost-effective extraction methods without compromising quality are desirable.

Validation with clinical samples

Clinical relevance: The cfDNA extraction method using clinical samples should be validated to ensure its suitability for diagnostic or research purposes.

User-friendliness

Ease of use: Assess the simplicity and user-friendliness of the extraction protocol to minimize variability between operators.

Automation potential: Automation of the extraction process can increase throughput, reduce hands-on time, and minimize variability introduced by manual handling. Consideration of the automation potential of extraction methods may be important for high-throughput applications.

Regularly monitoring these parameters and optimizing the cfDNA extraction process based on the specific requirements of your study or application can help ensure reliable and consistent results. Additionally, staying informed about advancements in cfDNA extraction techniques may lead to improvements in efficiency and sensitivity.

Fragment size distribution

The size distribution of cfDNA fragments can impact its usability in various downstream applications. Some applications may require specific fragment sizes, so ensuring the extraction method yields cfDNA with appropriate fragment lengths is important.

Reproducibility

Consistency in cfDNA extraction across multiple samples and experiments is essential for reliable results. Extraction methods should be robust and reproducible to minimize variability between samples and experiments.

Efficiency

Extraction methods should efficiently recover cfDNA from various sample types (e.g., blood plasma, serum, urine) and volumes. High extraction efficiency reduces the risk of losing valuable cfDNA molecules during the extraction process.

Compatibility with downstream applications

The extracted cfDNA should be compatible with the intended downstream analysis methods, such as NGS, PCR, digital PCR, or other molecular biology techniques. Compatibility ensures seamless transition from extraction to analysis.

Sample input requirements

Some extraction methods may have specific requirements regarding sample input volume, type, or quality. Understanding these requirements is crucial for selecting the appropriate extraction method for the available sample type and volume.

cfDNA solutions for NGS library preparation from IDT

IDT’s xGen™ product family includes the xGen cfDNA and FFPE DNA Library Prep Kit. This solution permits high library complexity from low-quality samples, providing highly complex variant ID from degraded and low-input research samples. Automation capabilities are built into the tool, with no expertise necessary.

The xGen cfDNA & FFPE DNA Library Prep Protocol takes about 4 hours and includes only four major steps, minimizing sample handling.

Learn more about xGen NGS library prep here. Archer™ LIQUIDPlex™ is a full solution with analysis for solid tumor use when needed.