How to isolate good quality nucleic acids
The extraction of RNA from viral samples is an important step for pathogen research. Viral nucleic acid extraction is the first step for downstream genomic analysis. Analysis of viruses in biological and environmental samples requires efficient methods for viral nucleic acids that are amenable to a variety of sample types. Successful genome sequencing of SARS-CoV-2 depends on the quality of the nucleic acids extracted from the primary sample. Each sample type has unique requirements for optimal nucleic acid extraction and isolation.
Different types of samples are used for SARS-CoV-2 testing including:
- Swabs (nasopharyngeal and oropharyngeal)
- Other samples – bronchoalveolar lavage (BAL) and other respiratory research samples, urine, whole blood, plasma, tissue, stool, cell-free body fluids, and even wastewater
Due to the delicate nature of RNA, the RNA purification process consists of a variety of unique challenges, one of which is ribonuclease (RNAse) contamination. RNases are abundant in the environment and even a trace amount of RNase contamination can sabotage RNA-based experiments. Several precautions such as the use of RNase-free reagents, dedicated pipettes, glassware, gloves, and working in an RNAse-free environment need to be followed to achieve a good RNA yield.
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Often minute amounts (low viral load per ml, typically 1000-5000 particles/ml) of viral RNA need careful extraction from samples such as tissues, nasopharyngeal/oropharyngeal swabs, sputum, blood, plasma, or other body fluids. Viral RNA might also be extracted from clinical samples, water or other environmental samples. Sometimes investigators might need to quantify the viral particles contaminating medicinal products such as vaccines. Since the viral load of these biological, medicinal, and environmental samples is usually exceptionally low, RNA isolation consists of two major steps: virus concentration followed by RNA extraction. Viral concentration is usually achieved by applying various precipitation, flocculation, and filtration techniques.
The Organic Extraction Method
The organic extraction method is the most tried-and-tested method for RNA extraction and removal of cellular proteins. Here, RNA isolation is achieved through organic extraction followed by RNA precipitation. This technique involves lysis or extraction in a monophasic solution of phenol and guanidine isothiocyanate. Chloroform is then added. The phenol-chloroform mixture is immiscible with water. Therefore, when centrifuged, the sample forms two distinct phases: the lower (organic) phase and phase interface contain denatured proteins, while the less-dense upper (aqueous) phase contains the RNA. The aqueous phase containing the RNA is carefully removed by pipetting, without touching the interface or the lower organic phase, as this can contaminate the sample. The RNA is then precipitated with isopropanol and then rehydrated for further analysis.
Organic extraction protocols are well-established and are useful for most sample types. Proteins are rapidly denatured, and RNA is quickly stabilised. The process is scalable and can be completed in 30-60 minutes. However, this method is not amenable to high-throughput processing and is difficult to automate. New users find the phase separation and careful pipetting of the aqueous phase challenging to master. A chemical fume hood is required due to the use of the hazardous chemicals, which then need to be disposed of appriopriatly. Manual handling of a large number of samples is cumbersome.
Figure 1 – Workflow of an organic extraction protocol
The Spin-Column Based Method
The easiest and safest method, readily available in a kit format, is the spin-column-based method. The binding element in spin-column systems is usually composed of glass particles or powder, silica matrices, diatomaceous earth, and ion exchange carriers. In this method, nucleic acid binding is optimised with specific buffer solutions and extremely precise pH and salt concentrations. Sample lysates are passed through the silica membrane using centrifugal force, with the RNA binding to the silica gel at the appropriate pH. The membrane containing residual proteins and salt is then washed to remove impurities, and the flow-through is discarded. RNA is subsequently eluted with RNase-free water.
Figure 2 – Diagram showing the steps for viral RNA isolation using a standard commercial kit
Column-based RNA extraction is one of the best techniques among the options available, playing a vital role in ion exchange methods, as it provides a robust stationary phase for rapid and reliable buffer exchange and thus nucleic acid extraction. This method is fast and reproducible, and its main drawback is the need for a small centrifuge. Vacuum-based systems can also be used in place of centrifugation to separate impurities. Researchers can also combine the organic extraction method with the spin column method for faster and greater RNA yield. This method is fast (20 minutes) and amenable to large-scale and high-throughput processing, including automated methods. Protein or DNA contamination is possible if the sample amount is large or remains incompletely homogenised or lysed. Incomplete lysis can also lead to low yields of viral RNA. Automation can be complex and expensive due to the need for setting up centrifugation or vacuum-based separation systems.
The Magnetic Bead-Based Method
The magnetic bead-based method relies on the use of magnetic beads and reagents optimised for RNA extraction. The beads have a paramagnetic core, usually coated with silica for nucleic acid binding. The sample is lysed in a buffer with RNase inhibitors and then incubated with magnetic beads, allowing the particles to bind RNA molecules. The magnetic beads can then be quickly collected by being placed in proximity to an external magnetic field. The supernatant is removed, and beads are subsequently washed in a suitable wash buffer with the removal of the magnetic field. This process can be easily repeated for multiple washes. The RNA is eluted from the magnetic beads with RNase-free water into a solution, and the supernatant (containing the pure RNA) can then be transferred.
Figure 3 – Schematic of RNA isolation using magnetic bead-based technology
The magnetic bead collection steps are simple and quick to perform. There is a reduced risk of clogging as no column is involved. This technique is the most amenable to scale-up, high-throughput separation, and automation. The clean-up is more effective due to the movement of the beads. However, viscous samples could impede the movement of the beads and, occasionally, the final sample may be contaminated with magnetic beads. A magnetic stand is required for manual separation and a magnetic particle handler for the automation of this process.
Automated viral nucleic acid purification systems
For fast, easy, and effective high-throughput sample processing, RNA extraction protocols are automated. Automated viral RNA extraction protocols for the silica plate-based protocol use a vacuum manifold to achieve buffer flow/wash and are available for a variety of liquid handlers. Automated viral RNA extraction protocol for magnetic bead protocol uses a magnetic comb which carries the separated nucleic acids from the lysis step through wash steps and finally, viral RNA is eluted into an elution buffer.
Table 1 – Summary of key differences between spin column-based and magnetic bead-based viral RNA isolation
|Organic solvent hazardous waste
|Phase separation difficult for new users
|Handling several samples is tedious. Centrifuge/vacuum required
|Magnetic stand required
|Concern of clogging
Precaution 1: Samples may be fresh or frozen, but if frozen, should not be thawed more than once. Repeated freeze-thawing of plasma samples will lead to reduced viral titers and should be avoided for optimal sensitivity. Cryoprecipitates accumulate when samples are subjected to repeated freeze-thaw cycles which may lead to clogging of the silica membrane used for purification.
Precaution 2: RNA extraction is a delicate process, as cells and the environment secrete high concentrations of enzymes that destroy nucleic acids, therefore, the process must be carried out in a careful and quick manner.
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A Practical Guide for SARS-CoV-2 Whole Genome Sequencing
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