This blog was written by guest writers Paraj Mandrekar (Technical Services Scientist 3) and Michelle Mandrekar, (Research Scientist 4).
Here are some designer’s notes comparing the Maxwell® RSC Blood DNA and the Maxwell® RSC simplyRNA kit chemistries for nucleic acid extraction.
The Maxwell RSC Blood DNA Kit and Maxwell RSC simplyRNA Blood Kit were both developed from the same non-silica-based purification chemistry and use the same underlying paramagnetic particle. This chemistry is characterized by an extreme binding capacity (the capacity of nucleic acid that can be bound on the particle), leading to both chemistries being capable of isolating large amounts of nucleic acid volumes and then eluting into relatively small volumes (50 µL). It is not unusual with either chemistry to have isolates that exceed 100 ng/µL. Although the chemistries have several similarities, there are some important distinctions between how the two chemistries were designed that influence which kit you choose for your nucleic acid extraction.
This blog is written by guest blogger, Heather Tomlinson, Director of Clinical Diagnostics at Promega.
Finding safe and effective treatments for human diseases takes time. Medication and diagnostic tests can take decades to discover, develop and prove safe and effective. In the United States, the FDA stands as the gold-standard gatekeeper to ensure that treatments and tests are reliable and safe. The time we wait in review and clearance means less risk of ineffective or unsafe treatments.
And yet, in a pandemic, we are behind before we even start the race to develop diagnostic tests, so critical for understanding how an infectious disease is spreading. That is when processes like the FDA’s fast track of Emergency Use Authorization (EUA) are critical. Such authorization allows scientists and clinicians to be nimble and provide the best possible test protocol as quickly as possible, with the understanding that these protocols will continue to be evaluated and improved as new information becomes available. The EUA focuses resources and accelerates reviews that keep science at the fore and gets us our best chance at staying safe and healing.
For scientists working around the clock, the FDA’s EUA process is ready to review and respond. Getting an EUA gives clinical labs a very specific and tested resource to guide them to the tools and tests to use in a crisis.
Typically the Centers for Disease Control (CDC) will develop the first test or protocol that receives FDA EUA in response to a crisis like a pandemic. For COVID-19 the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel received FDA EUA clearance in early February. This is the test protocol used by the public health labs that work with the CDC and test manufacturers around the world.
Throughout a crisis such as the current pandemic, scientists continually work to improve the testing protocols and add options to the EUA protocols. This enables more flexibility in the test protocols. Promega is fortunate to play a part of the CDC EUA equation for diagnostic testing. Our GoTaq® Probe 1-Step PRT-qPCR System is one of a few approved options for master mixes in the CDC qPCR diagnostic test, and now our medium-throughput Maxwell 48 Instrument and Maxwell Viral Total Nucleic Acid Purification Kit have been added to the CDC protocol as an option for the RNA isolation step as well. These additions to the CDC EUA means that laboratories have more resources at their disposal for the diagnostic testing which is so critical to effective pandemic response.
The Emergency Use Authorization provides the FDA guidance to strengthen our nation’s public health during emergencies, such as the current COVID-19 pandemic. The EUA allows continual improvement of an authorized protocol through the collaborative efforts scientists in all academia, government and industry to identify and qualify the most reliable technologies and systems, giving labs more flexibility as new products are added as options.
Dr. Tomlinson is the Director for the Global Clinical Diagnostics Strategic Business Unit at Promega Corporation with over 15 years of experience in clinical diagnostic test development. She is responsible for leading the team that drives strategy in the clinical market for Promega. Her background is in infectious disease diagnostic testing, with a focus on HIV drug resistance and evolution. Her recent work has been in oncology companion diagnostic test development. Heather has is an accomplished international presenter, delivering conference presentations in the United States, Europe, Asia, and Africa.
A protein first purified and sold by Promega almost four decades ago has emerged as a crucial tool in many COVID-19 testing workflows. RNasin® Ribonuclease Inhibitor was first released in 1982, only four years after the company was started. At that time, the entire Promega catalog fit on a single sheet of 8.5 × 11” paper, and RNasin was one of the first products to draw widespread attention to Promega. Today, the demand for this foundational product has skyrocketed as it supports labs responding to the COVID-19 pandemic.
What is RNasin® Ribonuclease Inhibitor?
RNA is notoriously vulnerable to contamination by RNases. These enzymes degrade RNA by breaking the phosphodiester bonds forming the backbone of the molecule. To say that RNases are everywhere is barely an exaggeration – almost every known organism produces some form of RNase, and they’re commonly found in all kinds of biological samples. They’re easily introduced into experimental systems, since even human skin secretes a form of RNase. Once they’re present, it’s very hard to get rid of them. Even an autoclave can’t inactivate RNases; the enzymes will refold and retain much of their original activity.
RNasin® Ribonuclease Inhibitor is a protein that has been shown to inhibit many common contaminating RNases, but without disrupting the activity of enzymes like reverse transcriptase that may be essential to an experiment. It works by binding to the RNase enzyme, prevent it from acting on RNA molecules. This is important for ensuring that RNA samples are intact before performing a complex assay.
This blog was written with much guidance from Jennifer Romanin, Senior Director IVD Operations and Global Service and Support, and Ron Wheeler, Senior Director, Quality Assurance and Regulatory Affairs at Promega.
A Trip Down Memory Lane
Back in the day when we all walked two miles uphill in the snow to get to our laboratories, RNA and DNA extraction were home-brew experiences. You made your own buffers, prepped your own columns and spent hours lysing cells, centrifuging samples, and collecting that fluorescing, ethidium bromide-stained band of RNA in the dark room from a tube suspended over a UV box. Just like master beer brewers tweak their protocols to produce better brews, you could tweak your methodology and become a “master isolater” of RNA. You might get mostly consistent results, but there was no guarantee that your protocol would work as well in the hands of a novice.
Enter the biotechnology companies with RNA and DNA isolation kits—kits and columns manufactured under highly controlled conditions delivering higher quality and reproducibility than your home-brew method. These systems have enabled us to design ever more sensitive downstream assays–assays that rely on high-quality input DNA and RNA, like RT-qPCR assays that can detect the presence of a specific RNA molecule on a swab containing only a few hundred cells. With these assays, contaminants from a home-brew isolation could result in false positives or false negatives or simply confused results. Reagents manufactured with pre-approved standard protocols in a highly controlled environment are critical for ultra sensitive tests and assays like the ones used to detect SARS-CoV-2 (the virus that causes COVID-19).
The Science of Manufacturing Tools for Scientists
There are several criteria that must be met if you are
producing systems that will be sent to different laboratories, used by
different people with variable skill sets, yet yield results that can be
compared from lab to lab.
Depending on your viewpoint, source of information and tolerance for risk, this can be a frightening time for persons all over the planet. The level of disruption to daily life that we’re all experiencing due to COVID-19 is unprecedented.
We are all either not working, working from home and away from our normal offices, or in some cases working many more hours to cover for sick coworkers and caring for SARS-CoV-2-infected persons.
But there is good news if you find that information is power. We hope that some information about the testing being used in the US for this novel coronavirus might be fuel for you, empowering in terms of information.
What is the Name of the Virus, and the Disease? Since this is a global pandemic, the World Health Organization was instrumental in naming the virus and disease. From this web page: the disease is called COVID-19.
The coronavirus responsible for this disease is SARS-CoV-2.
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”
Aberrant RNA binding protein (RBP) function has been implicated in a host of human diseases from various cancers, neurological disorders, and conditions related to muscular atrophy (1). Understanding RBP function requires not only a working knowledge of the protein proper, but accurate methods to identify RNA binding partners in vivo. Identification of RNA binding partners has historically been difficult, especially for RNA targets involved in nervous system disorders. Methods for finding targets have involved in vitro RNA selection or co-immunoprecipitation followed by gene chip analysis (2,3). These approaches came with some inhert limitations. The signal to noise ratio is low and the ability to differentiate between direct and indirect interactions is limited. Additionally, since the RNA-protein interactions are so complex, any of the in vitro methods may not be wholly predictive of true intracellular interactions.
In 2003, researchers at the Laboratory of Molecular Neuro-Oncology at Rockefeller University developed a method to purify protein-RNA complexes from mouse brain tissue that utilized ultraviolet cross-linking of RNA to their protein binding partners and immunoprecipitation of the cross-linked product (4). Further development of the technology has resulted in a streamlined protocol to perform high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP; 5). Continue reading “Mapping Protein-RNA Interactions in vivo Using the HITS-CLIP Method”
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