LABTips: RNA Extraction and Purification for COVID-19 Testing

RNA extraction and purification is a crucial step in RT-PCR testing for COVID-19. Isolating any viral RNA that may be present in a patient sample is more than just a routine task — the efficiency of the extraction method can impact whether enough viral RNA will be available to be amplified and detected.

Properly following RNA extraction protocols and utilizing appropriate extraction methods can help reduce the risk of false negative results. Knowing the factors that contribute to successful RNA purification and PCR analysis will also help you balance sensitivity needs with throughput needs, ultimately providing patients with the fast and accurate results they need to make important health and safety decisions.

Consider these tips to optimize your RNA extraction and purification process for COVID-19 testing during the ongoing pandemic:

1. Use viral RNA loads to guide RNA recovery

One major factor that will affect the amount of RNA you will be able to extract if the virus is present is the differing viral loads typically found in different sample types and at different points in the progression of the COVID-19 disease.While you can’t typically know the time period since a person was exposed or infected, or control the exact types of samples coming into your lab, understanding the differences between different specimen types can give you an idea of how much viral RNA you can expect to be present in a sample if a person is infected.

Several studies have been done on the positive test rates for different sample types taken from infected patients to determine where the highest viral load may be available for detection.¹ Current research suggests that lower respiratory tract samples, such as bronchoalveolar lavage fluid and sputum—which are generally collected in a hospital setting—are the most likely to produce a positive result (least likely to produce a false negative) in an infected person. Nasopharyngeal (NP) swabs, which can be collected outside of a clinical setting and from asymptomatic individuals, are considered to be the best upper respiratory sample for receiving an accurate result.

Consider the types of samples your lab accepts and how often you see each sample type. NP swabs are typically the most common sample type, but some labs may receive higher volumes of lower respiratory samples from hospitals, or test higher volumes of self-collected saliva samples. Understanding the dynamics of viral load and shedding from different collection areas can help you ensure your extraction method is optimized for sufficient RNA recovery from the samples you encounter.

Beyond viral load variations, certain sample types. such as sputum. may be especially viscous, reducing the efficacy of some extraction reagents and increasing the likelihood of pipetting errors unless properly homogenized and liquified prior to RNA isolation and purification.² The CDC offers recommendations for preparation of sputum samples using a dithiothreitol solution.  

2. Compare and contrast methods to suit your sample and throughput needs

There are a number of different kits, machines and methods available specifically for isolation and purification of SARS-CoV-2 RNA, but not every method is one-size-fits-all. In general, RNA extraction techniques fall into three main categories: organic extraction, spin-column extraction and magnetic bead extraction.  Each of these major categories has its own advantages and disadvantages, and exact procedures may vary from kit to kit or lab to lab. While organic extraction has been considered a “gold standard” for RNA extraction, the unprecedented demands of the COVID-19 pandemic have made spin column and magnetic bead extraction methods more desirable due to their relative speed and ability to be automated.

Knowing the advantages and potential pitfalls of each method is important both for choosing methods to implement and for working more efficiently with the methods available at your lab. Spin column extraction is considered to be a convenient and easy-to-perform method that can be completed fairly quickly even when performed manually. However, it is more difficult to automate due to the need for a complex and costly centrifuge or vacuum setup, and the column's silica membrane presents the risk of clogging, which can result in low RNA yield. The use of silica-coated magnetic particles has no risk of clogging, can easily be automated for high-throughput processing and is highly efficient for RNA recovery and purification. The downsides are the difficulty of performing this method manually, the risk of samples becoming contaminated with residual magnetic particles and the fact that more viscous samples can impede the movement of the beads.^{3, 4}

Be attentive to the specific challenges for each method in order to prevent these problems and troubleshoot when they arise. You should also keep the differences between these methods in mind if you’re looking to increase your throughput or add more automation to your workflow.

3. Prevent degradation before, during and after extraction

The fragile nature of RNA makes it especially important to handle specimens, eluted RNA and each component of the extraction process with care to avoid degradation. RNase contamination and improper temperature conditions are two of the biggest culprits when it comes to RNA degradation.

Every effort should be made to maintain an RNase-free environment when working with samples for RT-PCR testing, and anything that comes in contact with the sample should be considered a potential source of contamination, from equipment and consumables (like pipette tips) to water and reagents, to workspace surfaces as well as your own skin.^5,6

Using pretreated RNase-free certified products is one way to reduce contamination, but keep in mind that these products can still end up contaminated if proper precautions are not taken. RNase-free consumables and solutions should be properly stored and sealed before use, and gloves should be worn at all times. Gloves should always be changed after coming in contact with potential sources of contamination, like door handles or lab equipment that has not been decontaminated. Use specialized RNA decontaminant solutions to clean surfaces, equipment and labware, but ensure the decontaminant you use won’t interfere with your reagents (ex. DEPC treatment interferes with Tris reagents). RNAse inhibitors can also be used to further protect RNA from damage.

To prevent degradation during storage, specimens should be refrigerated at 2-8 degrees Celsius for up to 72 hours after collection, or kept at -70 degrees Celsius or lower if extraction is not completed within 72 hours. Samples should be kept on a cold block or on ice while being prepared, and isolated RNA should be stored at -70 degrees Celsius or lower.⁷

4. Prepare for supply chain disruptions

The COVID-19 pandemic has strained supply chains in countless industries, and diagnostic supplies are certainly no exception. Last spring, severe shortages of RNA extraction kits led to testing backlogs and spurred clinicians to request donations from research labs. Even with vaccines now available and many areas recovering economically more than a year after the crisis began, do not let your guard down—expect that the availability of RNA extraction kits and reagents may not always be consistent.

Potential alternatives to commercial kits have been published to help diagnostic labs continue to operate in case of a supply shortage, including optimized manual protocols and phenol/guanidine-based organic extraction methods.⁸ In addition to following the research dedicated to these alternative methods, having a full understanding the principles of lysis, isolation and purification, and the reactions and reagents involved in each step, will also help you adapt your methods should you run short of a specific resource.

The unpredictable circumstances of the pandemic show why RNA extraction and purification for diagnostic testing is more than just a routine procedure. Performing this task mindfully will impact the success of your downstream analysis and prepare you for any challenges that may arise during this crucial process.

References

1. Bwire, GM, Majigo, MV, Njiro, BJ, Mawazo, A. Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis. J Med Virol. 2021; 93: 719– 725. https://doi.org/10.1002/jmv.26349
2. Peng J, Lu Y, Song J, Vallance BA, Jacobson K, Yu HB and Sun Z (2020) Direct Clinical Evidence Recommending the Use of Proteinase K or Dithiothreitol to Pretreat Sputum for Detection of SARS-CoV-2. Front. Med. 7:549860. doi: 10.3389/fmed.2020.549860 
3. "Viral RNA Isolation Methods Reviewed: Spin vs. Magnetic," kbDNA, Inc. https://www.kbdna.com/publishinglab/viral-RNA-isolation-methods-reviewed-spin-vs-magnetic 
4. Yu, C.Y.; Chan, K.G.; Yean, C.Y.; Ang, G.Y. Nucleic Acid-Based Diagnostic Tests for the Detection SARS-CoV-2: An Update. Diagnostics 2021, 11, 53. https://doi.org/10.3390/diagnostics11010053  
5. "The Basics: RNase Control," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/the-basics-rnase-control.html 
6. "Avoiding Ribonuclease Contamination," New England BioLabs Inc. https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination
7. CDC. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel SOP# CDC-006-00019, 2020. https://www.fda.gov/media/134922/download
8. C. Ambrosi, C. Prezioso, P. Checconi, D. Scribano, M. Sarshar, M. Capannari, C. Tomino, M. Fini, E. Garaci, A.T. Palamara, G. De Chiara, D. Limongi, SARS-CoV-2: Comparative analysis of different RNA extraction methods, Journal of Virological Methods. 287 (2021) 114008. https://doi.org/10.1016/j.jviromet.2020.114008.