Using molecular biology and biochemistry to detect blood doping

Monitoring the use of performance-enhancing substances among athletes is complex and the requirements for tests and assays that detect use of such substances have changed significantly over the last few decades.

The haematological (blood) module of Athlete Biological Passport was adopted December 1, 2009 (ABP) by the World Anti-Doping Agency. The module sets out standard protocols to monitor doping of professional athletes by looking at changes in biological parameters, without relying on the detection of illegal compounds in body fluids. Such biological methods eliminate the need to develop and validate a test to detect every new compound that can be used for doping. The current version of the ABP, adopted in 2014, also adds monitoring of certain steroid use indicators from urine samples.

Blood doping which aims at increasing red blood cells so that more oxygen can be transported to muscles to increase stamina or performance is particularly difficult to detect. There are typically three ways that it is accomplished: use of erythropoietin (EPO) or synthetic oxygen carriers and blood transfusions. While transfusions of large volumes of blood or use of EPO can be detected, microdosing EPO or transfusing smaller volumes of packed red blood cells is much harder to detect.

Nicolas Leuenberger and colleagues at the Swiss Laboratory for Doping Analysis have developed a method to detect blood doping. In addition to addressing the detection of blood doping, his laboratory is also concerned about easing the transport and storage requirements for samples and ensuring that sample collection does not adversely affect athlete performance.

Improving Collection and Storage of Blood Samples

Because sample collection and storage are so critical to accurate test results, any new assays developed to detect blood doping benefit from ease of collection and storage. The Leuenberger laboratory investigated the use of the TAP™ Push Button collection device, which is billed as a simple method for blood collection that is easy to use and eliminates the need for painful needle sticks or finger pricks that can affect athlete performance. After TAP collection, 20µl of blood from the device was placed on to filter paper and dried (dried blood samples; DBS), which are much easier to store and transport from collection site to laboratory.

An RNA Biomarker for Blood Doping

Blood withdrawal and autologous transfusion or recombinant human EPO injection stimulate erythropoiesis and immature red blood cells can be distinguished based on their gene expression profiles. One of the genes that is expressed by immature red blood cells is aminoleuvulinate synthase 2, a gene that encodes an enzyme ALAS2 involved in the synthesis of heme, a pathway active during RBC maturation. RNA transcripts are unstable and tend to degrade rapidly, so isolating linear RNA transcripts from a collected sample can be difficult. However circular RNAs (circRNAs) are a class of RNA molecule produced by the backsplicing of pre-mRNAs that are high in abundance, quite stable and maintain cell-type specific expression. The Leuenberger laboratory developed a method for measuring the linear and circular forms of ALAS2 RNA in DBS to monitor erythropoiesis.

One of the greatest challenges in developing this protocol was achieving efficient RNA extraction from only 20ul of dried blood. Leuenberger and his colleagues adopted a two-step purification; beginning with a phenol:chloroform extraction on the DBS followed by a further purification on the Maxwell® RSC automated instrument, using the Maxwell RSC miRNA Serum and Plasma kit. Switching from a manual to an automated method for the second step was crucial. It reduced chances of contamination as well reduced pipetting errors, without compromising good quality and yield of RNA therefore contributing to assay reproducibility. To normalize volumes within the blood spot, the protocol uses RNA produced by housekeeping genes. The work to automate the assay has been published in Bioanalysis.

What’s Next

This protocol is being tested to see if microdosing of EPO or small transfusions can also be detected by monitoring ALAS2 RNA expression in DBS. The Swiss laboratory of Doping Analysis is also in the process of developing a method to detect gene doping by isolating plasmid DNA from whole blood samples, using the Maxwell RSC.

Additionally, the collection and storage methods used have implications for the clinic, especially for patients that need routine blood monitoring. The ability to isolate circular RNAs shows promise in forensic applications to identify body fluids. 

Promega Leverages Long-Time Experience in MSI Detection with European Launch of CE-Marked IVD Assay for Microsatellite Instability

The genetic abnormality called microsatellite instability, or MSI, has been linked to cancer since its discovery in 1993 (1). MSI is the accumulation of insertion or deletion errors at microsatellite repeat sequences in cancer cells and results from a functional deficiency within one or more major DNA mismatch repair proteins (dMMR).  This deficiency, and the resulting genetic instability, is closely related to the carcinogenicity of tumors (2).

Historically MSI has been used to screen for Lynch Syndrome, a dominant hereditary cancer propensity. More recently, tumors with deficient MMR function have been identified as being more likely to respond to immune checkpoint inhibitor (ICI) therapies (3.). Because MSI can be the first evidence of an MMR deficiency, MSI-High status is predictive of a positive response to immunotherapies such as ICI therapies. (3).

Learn more about MSI in this short animation.
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38 Years After First Release, RNasin Protects COVID-19 Tests

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.

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RT-qPCR and qPCR Assays—Detecting Viruses and Beyond

We have all been hearing a lot about RT-PCR, rRT-PCR and RT-qPCR lately, and for good reason. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) is the technique used in by the Center for Disease Control (CDC) to test for COVID-19. Real-time RT-PCR, or quantitative RT-PCR (RT-qPCR)*, is a specialized PCR technique that visualizes amplification of the target sequesnce as it happens (in real time) and allows you to measure the amount of starting target material in your reaction. You can read more about the basics of this technique, and watch a webinar here. For more about RT-PCR for COVID-19 testing, read this blog.

Both qPCR and RT-qPCR are powerful tools for scientist to have at their disposal. These fundamental techniques are used to study biological process in a wide range of areas. Over the decades, Promega has supported researchers with RT-qPCR and qPCR reagents and systems to study everything from from diseases like COVID-19 and cancer to viruses in elephants and the circadian rhythm of krill.  

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Testing for COVID-19: How it Works

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.

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Researching the Researcher: Abbeah Navasca, 2019 Real-Time PCR Grant Winner

The three winners of the 2019 Real-Time PCR Grants have been hard at work in the six months since receiving their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.

Abbeah Navasca is a plant pathology researcher with the Tagum Agricultural Development Company, Inc. (TADECO*, Philippines). She is developing treatments for viral infections that affect one of Philippines’ largest and most valuable agricultural exports: bananas. As a result of the qPCR grant, she and two of her colleagues were able to participate in sample preparation and analysis workshops with Promega Technical Services experts in Singapore. During her visit, the team worked through strategies for plant sample preparation and amplified those samples with the GoTaq® 1-Step RT-qPCR System. We had a chance to ask her more before she headed back to her lab.

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Investigation of Remdesivir as a Possible Treatment for SARS-2-CoV (2019-nCoV)

Remdesivir (RDV or GS-5734) was used in the treatment of the first case of the SARS-CoV-2 (formerly 2019-nCoV ) in the United States (1). RDV is not an approved drug in any country but has been requested by a number of agencies worldwide to help combat the SARS-CoV-2 virus (2). RDV is an adenine nucleotide monophosphate analog demonstrated to inhibit Ebola virus replication (3). RDV is bioactivated to the triphosphate form within cells and acts as an alternative substrate for the replication-necessary RNA dependent RNA polymerase (RdRp). Incorporation of the analog results in early termination of the primer extension product resulting in the inhibition.

 Note the spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. In this view, the protein particles E, S, M, and HE, also located on the outer surface of the particle, have all been labeled as well. A novel coronavirus virus was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China in 2019.
This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM CDC

Why all the interest in RDV as a treatment for SARS-CoV-2 ? Much of the interest in RDV is due to a series of studies performed by collaborating groups at the University of North Carolina Chapel Hill (Ralph S. Baric’s lab) and Vanderbilit University Medical Center (Mark R. Denison’s lab) in collaboration with Gilead Sciences. 

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Researching the Researchers: Alberto Biscontin

2019 Real-Time PCR Grant

The three 2019 Real-Time PCR Grant Winners have been hard at work in the six months since winning their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.

One of the 2019 winners, Alberto Biscontin (University of Padova, Italy), performs research in the fields of Neurogenetics and Chronobiology. He is looking to shed greater light on the circadian rhythms of the Antarctic krill. Alberto published his most recent analysis in Nature and GoTaq® qPCR Master Mix helped him validate expression of genes for his study.

His qPCR data showed support for internal mechanisms that not only support daily living but also clarified the overwintering process of the krill. Now that Alberto has sized up some zooplankton, we asked him to share a little more about himself and his research:

Q: How long have you been a researcher?
A: I have been a researcher since 2012.

Q: How did you decide to research Antarctic krill?
A: In 2013, I had the opportunity to join the international Antarctic research program PolarTime. [It] brought together eight research groups with different scientific expertise to study seasonal and daily rhythms in the Antarctic krill Euphausia superba.

Q: When you are not busy at the bench, what do you like to do?
A: Traveling. I love strolling through open-air markets.

Q: Are there any tips or tricks you have learned that make your job easier?
A: You can easily switch from a classic RT-PCR protocol to a cheaper and faster One-step protocol using the same primers and temperatures.

Q: What comes next?
A: I would like to characterize the clock machinery of other polar organisms to understand whether high latitude clocks have developed similar strategies to cope with [the] polar environment. Moreover, a better understanding of marine circadian clocks could help to shed light on the evolution of the animal circadian machinery.

You can find Alberto’s most recent publication in Nature Scientific Reports. The 2020 Real-Time PCR Grant will be coming soon. For more information on the 2019 winners and information on the 2020 Grant, visit the Real-Time Grant web page. Be sure to follow us on social media for the most up-to-date information regarding the 2020 Grant, including application deadlines and winner notifications!

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One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems. This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The insert is directly ligated to the T-tailed plasmid vector with T4 DNA ligase. The insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the multiple cloning region of the T vector.

Proofreading polymerases like Pfu do not add “A” overhangs so PCR products generated with these polymerases are blunt-ended. In a previous blog, we discussed a simple method for adding an A-tail to any blunt-ended DNA fragment to enable T-vector cloning. Below, we think about the next step: Ligation.

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CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology

Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.

Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.

Continue reading “CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology”