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How to Automate Viral Nucleic Acid Extraction from Plasma, Saliva, Feces and Swabs

Automated extraction of viral nucleic acids from viscous samples, such as serum, plasma, saliva, feces or swabs, can be challenging. Learn more about the challenges and ways to overcome them in this article.

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Viral Nucleic Acid Extraction Workflow

Automated extraction of viral nucleic acids from serum, plasma, saliva, feces or swabs involves a few steps:

  1. Sample Preparation: 
    • Serum and plasma: Centrifugation of the sample is needed to remove any cellular debris or contaminants. Serum and plasma tend to be viscous, therefore difficult to accurately pipette and transfer.
    • Saliva: Saliva shares many of the properties of serum and plasma; however, the viscosity can be higher, making it more difficult to manage.
    • Feces: Fecal samples generally require homogenization and debris removal, and they can be high in biomass and inhibitors.
  2. Lysis: The sample is lysed using a lysis buffer that contains salts (e.g., guanidine thiocyanate), detergents (e.g., SDS) and other reagents that help denature proteins and release nucleic acids from the virus. Proteinase K can improve extraction efficiency by degrading proteins and improving the lysis of viral particles.
  3. Binding: The lysed sample is mixed with a binding buffer. Silica magnetic particles, cellulose or other coatings that facilitate selective nucleic acid binding are often included.
  4. Washing: Bound RNA and DNA are washed several times to remove any impurities.
  5. Elution: RNA and DNA are eluted from the silica particles using an elution buffer.

Automating this process can help to increase efficiency and reduce errors. However, there are a few challenges that can arise when automating this process. We will discuss the challenges of various samples types and how to overcome them here.

Viscous Sample Preparation (Serum, Plasma, Saliva, Blood)

Automation requires precise handling of samples, which can be difficult with plasma samples that are viscous and contain clots or other contaminants. Centrifugation and filtration can help solve this.

An automated instrument can incorporate a centrifuge for preparation of plasma or serum from whole blood. However, more expensive and complex camera systems are required to precisely identify the separation layers. Without a camera system, the instrument may struggle to accurately pinpoint the starting point of the plasma layer, potentially leading to contamination by cellular debris. Depending on the required plasma volume, it may be possible to use a fixed height for pipetting from the top, while allowing for variations between samples. For example, if you are working with less than 1ml of serum or plasma, there should be adequate space within a 10ml blood collection tube to accommodate the cellular and debris fraction post-centrifugation.

Plasma samples can contain clots, fibrin and other debris that can clog pipette tips or other automation equipment. One solution is to use specialized tips or other equipment that can handle viscous samples without clogging. For example, some pipette tips are designed with wider openings or internal structures that reduce clogging and improve sample flow. The wide bore tips are also helpful for dispensing viscous liquids, although they are generally not required. With proper liquid class development, plasma can be dispensed with normal tips. 

A good starting point for plasma and serum is a “blood” liquid class definition, which most companies provide as a default setting. Learn more about proper liquid class development in the "How to Build Liquid Classes" guide.

Saliva is highly viscous, may contain food particles, possesses a substantial buffering capacity, and may contain elevated levels of calcium phosphate salts. Therefore, the use of proteinase K is recommended when working with such samples. Centrifugation of saliva samples following the lysis step is also beneficial to eliminate debris, including food particles. In addition, sample dilution facilitates mixing during the binding step. The collection reagents themselves can contribute to dilution, making the samples more manageable downstream. Given the substantial viscosity of saliva, the use of wide-bore pipettes, similar to those employed for serum and plasma, can be advantageous. Learn more about automated liquid transfers of saliva in the "How to Build Liquid Classes" guide.

Viscous Sample Nucleic Acid Extraction 

Ensure you include a manual control when developing or troubleshooting a method. The manual control serves as the best benchmark for identifying problems. You can also monitor extraction efficiency with an internal control during the extraction process to confirm that everything is working correctly. Using only an internal control would not distinguish whether the automated method is faulty or the chemistry being used is inadequately extracting the nucleic acids. It is important to document and record all steps taken during troubleshooting to ensure that the same issues do not reoccur. By taking these steps, you can identify the root cause of low nucleic acid yield during automated DNA or RNA extraction from saliva, serum, plasma or other viscous biological liquids. This allows you to take appropriate measures to optimize the extraction process and improve DNA or RNA yield. Learn more about optimization in the "How to Automate Nucleic Acid Extraction" guide.

Proper mixing is crucial for efficient automated nucleic acid extraction, and this can be difficult to achieve with automated systems. It is arguably one of the most important steps when automating extraction as it is the primary factor affecting binding, washing and elution, assuming the kit meets your requirements manually.

The most important factor here is to visually ensure that a complete vortex forms and particles are fully suspended, as this is the most reliable indicator of efficient mixing.

Pipette mixing can also serve as a means to ensure proper mixing; however, it is the least efficient method. By repeatedly pipetting the sample and lysis buffer, you can achieve homogenization and even distribution of the lysis buffer within the sample. While this technique can effectively disperse clumps during washing steps, it proves highly inefficient for binding steps, which typically require 10–20 minutes.

It is important to ensure uniform distribution of lysis buffer throughout the sample. Proper mixing is especially important for viscous samples such as plasma, which can be difficult to handle and require more thorough mixing to ensure that the lysing buffer mixes uniformly. Insufficient mixing can lead to incomplete lysis of the viral particles, resulting in reduced DNA/RNA yields and compromised sensitivity of downstream assays.

During the binding step, mixing is necessary to ensure that the nucleic acids come in contact with the magnetic particles. To confirm proper mixing, visually inspect that the magnetic particles remain suspended as complete particle suspension is necessary for the wash buffer to be effective. Any remaining clumps will not be effectively washed (proteins are often the cause of particle clumping).

While the above principles apply to serum and plasma, you can learn more details in the "How to Automate Nucleic Acid Extraction" guide. In this comprehensive resource, you will find a video tutorial, guidance on experimental design for method development, and a troubleshooting guide that addresses each step.

Saliva samples may contain lower levels of viral RNA compared to other sample types, posing challenges for viral detection and downstream analyses. To maximize DNA/RNA yield, it may be required to optimize the extraction process, including adjustments to the lysis buffer and other components. In many cases, the observed reduction in yield compared to a functional manual control is attributed to inefficient binding, although poor elution may also be a contributing factor. Ensure that the correct volume of saliva sample is used during the extraction process, as using too little sample can result in diminished yield.

Saliva samples can be more variable than other sample types, with differences in viscosity, pH, and other factors that can affect the efficiency of nucleic acid extraction. When developing a method, try collecting a diverse set of samples to ensure your process can robustly handle the various range of samples.

Proteinase K digestions can help increase yield, improve lysis of capsid coated viral particles, and reduce protein burden in saliva, serum and other high-biomass biological samples. These viscous samples are protein-rich, leading to potential bead clumping issues. One solution is to include an additional wash step to reduce contamination and improve DNA/RNA purity. It is critical that the magnetic particles resuspend adequately during the wash steps. Adding additional wash steps can help if you encounter difficulties in particle resuspension.

Fecal Sample Preparation

Extracting viral DNA and RNA from fecal samples can be challenging due to the presence of inhibitors and other contaminants that can interfere with downstream applications. Here are the common steps involved in automated nucleic acid extraction from fecal samples using Promega kits, along with potential problems that may arise during the process.

  1. Sample Collection: Fecal samples can be collected using various methods, such as sterile swabs or fecal collection tubes. The variability of sample collection can lead to differences in DNA/RNA yield and quality.
  2. Homogenization: Fecal samples are typically homogenized in a lysis buffer to break open viral particles and release nucleic acids. Incomplete homogenization is a potential issue that can lead to reduced DNA/RNA yield and quality.

When performing fecal extractions, less is more. Fecal samples often have high biomass and can contain PCR inhibitors at high concentrations. Highly-concentrated fecal samples are also difficult to pipette. For automation, it's preferred to use 10–20% mass-to-volume solutions of feces or under 50mg of dry mass for a "miniprep scale" to ensure consistency. Although yields may be lower, maintaining around 50mg per extraction can significantly improve consistency, even though the kit may support up to 200mg per extraction. Particle movers like the Maxwell® RSC Instrument or Kingfisher™ System are effective for higher biomass samples, as they eliminate the need to transfer the fecal suspension during the process.

Fecal samples are typically homogenized in a lysis buffer to break open the viral particles and release RNA. While physical disruption methods like bead beating are not needed to lyse viral particles, they can help break down fecal matter and increase overall extraction efficiency. Proteinase K can also boost extraction efficiency by degrading proteins and improving the lysis of viral particles.

PCR inhibitors, such as humic acids, can pose challenges for viral nucleic acid extraction from feces, although inhibitor concentration should be similar for automated or manual extraction. If your manual extraction control is working, but automated extraction isn't, this suggests an issue with the wash steps. Ensure particles fully suspend during the wash, and consider adding an additional wash step. If the manual control doesn't work either, try using alternative kits like the Maxwell® RSC Fecal Microbiome DNA Kit.

Swab Sample Preparation

Extraction from swabs like NP swabs or buccal swabs is generally straightforward, and the guidance provided for other sample types can address sample-specific challenges (e.g., fecal swabs). There is, however, a simple trick for streamlining the automation of swab tubes, which eliminates the need for manual removal of swabs. This is a technique known as the "Tip Dance".

The Tip Dance involves moving a pipette tip around the interior of a primary sample tube containing a swab. This motion leaves the swab behind after aspirating the sample from the swab tube. With this technique, the pipette tip can efficiently aspirate the required sample volume and avoid the natural adherence of the sample swab to the pipette tip. See how the Tip Dance is implemented in this video.

Purification

To learn about purification, refer to the "How to Automate Nucleic Acid Extraction" resource. In this comprehensive resource, you will find a video tutorial, guidance on experimental design for method development, and a troubleshooting guide that addresses each step. With these resources, you will learn how to efficiently automate viral nucleic acid extraction from feces, saliva, blood, serum, plasma, and swabs.

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