Transcription is the production of RNA from a DNA sequence. It’s a necessary life process in most cells. Transcription performed in vitro is also a valuable technique for research applications—from gene expression studies to the development of RNA virus vaccines.
During transcription, the DNA sequence is read by RNA polymerase to produce a complimentary, antiparallel RNA strand. This RNA strand is called a primary transcript, often referred to as an RNA transcript. In vitro transcription is a convenient method for generating RNA in a controlled environment outside of a cell.
In vitro transcription offers flexibility when choosing a DNA template, with a few requirements. The template must be purified, linear, and include a double stranded promoter region. Acceptable template types are plasmids or cloning vectors, PCR products, synthetic oligos (oligonucleotides), and cDNA (complimentary DNA).
In vitro transcription is used for production of large amounts of RNA transcripts for use in many applications including gene expression studies, RNA interference studies (RNAi), generation of guide RNA (gRNA) for use in CRISPR, creation of RNA standards for quantification of results in reverse-transcription quantitative PCR (RT-qPCR), studies of RNA structure and function, labeling of RNA probes for blotting and hybridization or for RNA:protein interaction studies, and preparation of specific cDNA libraries, just to name a few!
In vitro transcription can also be applied in general virology to study the effects of an RNA virus on a cell or an organism, and in development and production of RNA therapeutics and RNA virus vaccines. The large quantity of viral RNA produced through in vitro transcription can be used as inoculation material for viral infection studies. Viral mRNA transcripts, typically coding for a disease-specific antigen, can be quickly created through in vitro transcription, and used in the production of vaccines and therapeutics.
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.
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.
Want to know more about how the Maxwell® RSC can give you the freedom to focus on the work that interests you the most? To learn more, click here.
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).
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.
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.
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.
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.
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.
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.
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!
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.
By clicking “Accept All”, you consent to the use of ALL the cookies. However you may visit Cookie Settings to provide a controlled consent.
If you are located in the EEA, the United Kingdom, or Switzerland, you can change your settings at any time by clicking Manage Cookie Consent in the footer of our website.
Necessary cookies are absolutely essential for the website to function properly. These cookies ensure basic functionalities and security features of the website, anonymously.
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertisement".
This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
6 months 2 days
This cookie is set by the provider Media.net. This cookie is used to check the status whether the user has accepted the cookie consent box. It also helps in not showing the cookie consent box upon re-entry to the website.
This cookie is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics the number of visitors, bounce rate, traffic source, etc.
This cookie is associated with Sitecore content and personalization. This cookie is used to identify the repeat visit from a single user. Sitecore will send a persistent session cookie to the web client.
This domain of this cookie is owned by Vimeo. This cookie is used by vimeo to collect tracking information. It sets a unique ID to embed videos to the website.
1 month 18 hours 24 minutes
This cookie is used to calculate unique devices accessing the website.
This cookie is installed by Google Analytics. The cookie is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. The cookies store information anonymously and assign a randomly generated number to identify unique visitors.
This cookie is installed by Google Analytics. The cookie is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form.
Advertisement cookies are used to provide visitors with relevant ads and marketing campaigns. These cookies track visitors across websites and collect information to provide customized ads.
1 year 24 days
Used by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
This cookie is set by doubleclick.net. The purpose of the cookie is to determine if the user's browser supports cookies.
5 months 27 days
This cookie is set by Youtube. Used to track the information of the embedded YouTube videos on a website.
Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors.
This cookies is set by Youtube and is used to track the views of embedded videos.
This is a pattern type cookie set by Google Analytics, where the pattern element on the name contains the unique identity number of the account or website it relates to. It appears to be a variation of the _gat cookie which is used to limit the amount of data recorded by Google on high traffic volume websites.