Coronavirus, RNA extraction, and magnetic beads
The last few weeks the coronavirus and the COVID19 epidemic have occupied a central place in our lives. One of the basic tools of coping with an illness is the ability to identify and treat or isolate those that are sick. However, the coronavirus presents a serious problem as those that are infected begin to infect others before they develop symptoms and realize that they are sick. Moreover, some do not develop any disease symptoms and will not realize that they are “sick” at any point (and are called asymptomatic).
The primary test for this disease is based on the detection of the virus in the subject's respiratory system. The test is based on familiarity with the virus genetic material and examines whether there are unique virus sequences in the sample. That is, sequences of DNA/RNA letters that appear only in a COVID19 virus gene and in other viruses or in human genes. If there are such sequences, the answer is positive. The coronavirus is an RNA virus, meaning that its genetic information is stored as a RNA molecule. Thus, the test also involves identifying unique RNA sequences of viral origin.
Briefly, the test consists of several steps:
Using a specialized swab (nasopharyngeal swab) one scrapes cells and mucus from the throat and nose, areas where the virus appears in the early stages of the disease (later it will spread to other organs).
The sample from the swab is mixed with a lysis buffer that neutralizes the virus and releases the RNA molecules into the solution and protects them from degradation by RNases (proteins that break RNA molecules).
To perform the molecular test, the RNA is then isolated from the solution, since it contains cellular (and viral) debris as well as components from the solution which interfere with the test process.
The test is performed by a quantitative real-time PCR (Polymerase Chain Reaction) instrument that detects with high sensitivity whether a preselected viral sequence is present in the sample.
Our innovation is in the third phase of RNA extraction (or clean up). There are many ways to extract RNA (and / or DNA) from a solution. When performing medical diagnosis, accuracy and reliability are most important. For this reason diagnostic labs work with "closed" systems that typically include a dedicated device that is loaded with samples and consumables made specifically for this purpose. The advantage of these methods is that the user (in this case the test lab) knows that the materials are in the right quantities and work in a way that was designed without human error. The disadvantage of such an approach is the cost of consumables and the requirement to use consumables of a specific company (examples from day to day life - buying original printer ink cartridge or original espresso capsules).
The additional cost is usually worth it since it ensures accuracy, ease of operation, and low chance of human errors. However, these calculations break down during a pandemic. As the number of required tests becomes much larger, the cost of these kinds of tests can become a limiting factor. Access to proprietary consumables also becomes an issue. In normal times, companies have reasonable estimates of expected consumption and can meet the markets’ demand. However, the outbreak of the epidemic, in China, and then in neighboring countries such as Korea and Japan, led to a depletion of testing consumables. Now, with the transition to a global pandemic, testing companies are unable to meet huge demand from all around the world. The result is a global shortage, which limits test numbers exactly when they are most needed.
What does all this have to do with labs at the Life Sciences Institute? In our research we do a lot of genomic studies, and we often sequence RNA from small samples. This also means we have to deal with RNA extraction. About a month ago, when we realized that the RNA production phase might delay the rate of testing, we said "we have methods to do it easily." The answer we got was, "Don’t bother, there are reliable and working solutions." And so we moved on to thinking about a more ambitious challenge: how to test tens of thousands of samples at a time. We are still working on this challenge, and without going into too many details, part of the solution required "fishing" RNA from the harsh lysis buffer. We considered various solutions and to our delight some of them were found to be effective in capturing RNA directly from the solution. When we heard about the scarcity of consumables for RNA cleaning devices, we realized that we actually have a solution in hand. We chose a solution that seemed the simplest to implement.
What is the solution? It turns out to be one of the most useful tricks in genomics today. This is the use of microscopic beads (diameter of a micron, 1/1000 of a millimeter) whose core is paramagnetic (i.e., becomes a magnet when a magnetic field is applied on it). The outer shell of the bead consists of a negatively charged polymer. Using these beads is based on a neat trick. In the aqueous solution the charge of the bead repels nucleic acids (which are the building blocks of the RNA and are also negatively charged). However, when positive ions (e.g., salt) and a polymer called polyethylene glycol (PEG) are added to the solution, the situation changes. The PEG molecules capture the water molecules that retain the nucleic acids in the solution. On the other hand, the positive ions neutralize the negative charges. As a result, at the right concentration of salt and PEG, nucleic acids begin to aggregate to each other and also to the surface of the beads. Shortly afterwards most of the nucleic acids in the solution are immobilized on the beads. At this point, a strong magnet is used to pull the beads with the RNA on them to the side of the test tube. The beads and nucleic acids can now be isolated from the solution by removing the remaining solution. If we now add water to the test tube, we will reverse the equilibrium and the nucleic acids will dissolve in water, and we will get a solution containing only the nucleic acids.
To turn such a method into a routine tool in clinical diagnostic laboratories, they need to apply it on a large number of samples in a reliable manner. To achieve this it was crucial to automate the procedure on a robotic liquid handling device (or, more simply, arobot). This is a device that has the ability to work with 96 examples at the same time (see attached image). There were a number of challenges in automating all the steps of the protocol, but as it happens, we programmed a robot for these steps in a previous project in our lab. This allowed us, in just a few days, with the help of a volunteer who is an expert in programming such devices, to get a robust working system.
Why is the solution surprising? First, with the exception of the beads, the other materials (like the PEG) are cheap and accessible. It turns out that the beads themselves are not very expensive and we have obtained a sufficient amount for processing a large number of samples (over half a million). And so we have solved the dependence on reagents that have become a rare commodity. Second, we found that the proposed process is more efficient than the processes currently used. Using less than one-sixth of the original sample volume, we get the same amount of RNA. Additionally, the process is much faster. Finally, because the protocol is public and devoid of trade secrets, secret materials, and dedicated plastics, it can be implemented on a number of robotic devices found in research labs. The fact that we have been able to shorten a 2-4 hour step (depending on the type of device) to half an hour significantly increases the output of the diagnostic lab and allows for sufficient testing to deal with the epidemic.
To conclude, we are still striving to develop more efficient testing methods by replacing the measurement step, from PCR to DNA sequencing. Along the way, a group of researchers from the Institute of Life Sciences and the Center for Brain Sciences, laboratory managers, students and volunteers was built. Together with colleagues at the Medical School and Hadassah, we were able to make rapid progress in this direction. The combined effort of a large group of scientists from different backgrounds has allowed many directions to be tested at the same time, and so we have progressed an impressive amount in just a few weeks. Due to the ongoing epidemic, we split into small teams working in separate labs. Experiments on results and design of experiments are done by zoom and telephone.
I hope, in a time not far from now, we can all meet together and have a group photograph without fear of contagion. Until then, I hope that all of us will keep our safety rules and health.