Extraction of cfDNA
Extracting high-quality cell-free DNA (cfDNA) from blood requires careful consideration of several factors to ensure accurate and reliable downstream analysis. Here are some key considerations:
Blood Collection Tube and Anticoagulant
Choice of Tube: The type of blood collection tube significantly impacts cfDNA yield and quality. Tubes containing EDTA (ethylenediaminetetraacetic acid) are generally used for genetic testing as EDTA chelates magnesium ions, which are cofactors for DNases (enzymes that degrade DNA), thus minimizing cfDNA degradation and cellular DNA contamination. However if collecting in EDTA tubes samples would need to be processed within the hospital/site and ideally within 4-8 hours to avoid hemolysis and nucleated cell break-down which would negatively impact the downstream testing.
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Avoidance of Heparin: Heparin can interfere with certain downstream enzymatic reactions, such as PCR amplification, which are commonly used in cfDNA analysis. Therefore, heparinized tubes should generally be avoided.
Specialized cfDNA Tubes: Such as the Prenata Preserver tube have been developed and specifically designed for cfDNA preservation. These tubes contain additives that further stabilize cfDNA and prevent the release of cellular DNA from blood cells, allowing for longer storage times and potentially higher cfDNA yields.
Plasma Separation Timing and Protocol
Prompt Processing: To minimize the release of cellular DNA into the plasma due to cell lysis, blood samples should be processed to separate plasma as soon as possible, ideally within a few hours of collection. Delays can lead to significant contamination and inaccurate cfDNA quantification.
Centrifugation Protocol: A two-step centrifugation protocol is recommended. The first spin at a lower speed removes the majority of blood cells. The second spin at a higher speed helps to remove any remaining cellular debris and platelets, which can also release DNA. Standardized centrifugation speeds and durations are crucial for consistent results.
Plasma Storage Conditions
Temperature: If immediate cfDNA extraction is not possible, plasma should be stored at appropriate temperatures to prevent degradation. Short-term storage (up to 24-72 hours) is generally acceptable at 4°C. For longer storage periods, freezing plasma at -20°C or -80°C is recommended. Repeated freeze-thaw cycles should be avoided as they can damage cfDNA.
Aliquotting: To minimize the impact of freeze-thaw cycles, plasma samples should be aliquoted into smaller volumes before freezing. This allows for the use of only the required amount of plasma for each extraction.
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cfDNA Extraction Method
Silica-Based Membrane Kits: These are the most widely used methods for cfDNA extraction due to their efficiency, reliability, and scalability. They involve binding DNA to a silica membrane under specific buffer conditions, followed by washing steps to remove contaminants and elution of the purified cfDNA.
Magnetic Bead-Based Methods: These methods offer automation potential and can be particularly useful for high-throughput applications. DNA binds to magnetic beads, which are then separated using a magnet, washed, and the DNA is eluted.
Considerations for Method Selection: The choice of extraction method can influence the yield and size distribution of the extracted cfDNA. Factors to consider include the input plasma volume, the expected cfDNA concentration, the downstream application, and the required purity of the cfDNA.
Quality Control and Quantification
cfDNA Quantification: After extraction, it's essential to accurately quantify the cfDNA yield using methods like fluorometric assays (e.g., Qubit) or quantitative PCR (qPCR). This information is crucial for downstream analysis.
Size Distribution Analysis: Techniques like capillary electrophoresis can be used to assess the size profile of the extracted cfDNA. cfDNA typically shows a characteristic peak around 170 base pairs with smaller fragments, which can help in assessing the quality and integrity of the extracted DNA and identifying potential contamination with high molecular weight genomic DNA.
Assessment of Inhibitors: The presence of PCR inhibitors in the extracted cfDNA can affect downstream amplification. Assessing the presence of inhibitors and taking steps to remove them if necessary is important for reliable results.
Avoiding Contamination
Aseptic Techniques: Strict adherence to aseptic techniques during all steps of blood processing and cfDNA extraction is crucial to prevent contamination with exogenous DNA. This includes using sterile consumables, working in a clean environment, and wearing gloves.
When separating blood plasma from the double-spun blood tube its really important to be careful to remove the plasma without interacting with or interrupting the plasma-buffy layer interface as the buffy layer contains white bloods cells and would be a major source of contaminating high molecular weight DNA into the extraction.
Dedicated Workflows: Ideally, cfDNA extraction should be performed in a dedicated area separate from high molecular weight DNA work to minimize the risk of cross-contamination.
By carefully considering these factors, researchers and clinicians can optimize their protocols for cfDNA extraction from blood, leading to more accurate and reliable results for genetic analysis.
Follow this link to learn more about the Prenata extraction kits.