One of the most critical parts of a Next Generation Sequencing (NGS) workflow is library preparation and nearly all NGS library preparation methods use some type of size-selective purification. This process involves removing unwanted fragment sizes that will interfere with downstream library preparation steps, sequencing or analysis.
Different applications may involve removing undesired enzymes and buffers or removal of nucleotides, primers and adapters for NGS library or PCR sample cleanup. In dual size selection methods, large and small DNA fragments are removed to ensure optimal library sizing prior to final sequencing. In all cases, accurate size selection is key to obtaining optimal downstream performance and NGS sequencing results.
Current methods and chemistries for the purposes listed above have been in use for several years; however, they are utilized at the cost of performance and ease-of-use. Many library preparation methods involve serial purifications which can result in a loss of DNA. Current methods can result in as much as 20-30% loss with each purification step. Ultimately this may necessitate greater starting material, which may not be possible with limited, precious samples, or the incorporation of more PCR cycles which can result in sequencing bias. Sample-to-sample reproducibility is a daily challenge that is also regularly cited as an area for improvement in size-selection.
Welcome to the emerging frontier of immunometabolism. A decade ago, immunology and metabolism were seen as two distinct areas of study. However, we now know that specific metabolic activities are required for proper immune cell differentiation and function. In tumor microenvironments, immune cells may even alter their metabolism to compete with tumor cells for limiting nutrients.
Glucose metabolism in Naïve vs Effector T cells
What does your car and T cells have in common? They both shift gears! You can shift gears on your car to change the way the engine’s power is used to match driving conditions; when you’re going uphill, you switch to a higher gear. Similarly, when T cells are activated, they change the way they generate energy to match functional needs. This makes sense because activated T cells (known as effector T cells) require more energy and biomass to support growth, proliferation and effector functions.
While cars run on gas, the main fuel for T cells is glucose. Each glucose molecule is broken down into pyruvate while generating 2 ATP molecules. Naïve T cells completely oxidize pyruvate through oxidative phosphorylation to generate 36 ATPs per glucose molecule. However, when T cells are activated and become effector T cells, glycolysis is used to produce 2 ATPs per glucose molecule. Continue reading
Ribbon diagram of RNA’s biggest threat: a ribonuclease
RNA analysis from RT-pPCR to RNA-seq has become an increasingly important part of life science research as we seek to understand gene expression patterns, cell signaling and developmental events. To be successful at these RNA analysis steps, however, the upstream RNA purification needs to produce intact, high-quality product suitable for downstream work. Many RNA purification systems are available, ranging from high-throughput to manual using a variety of chemistries. You can purify RNA from FFPE or fresh mammalian tissues. How do you know which system to choose and when to use it? Our free webinar on August 11, The Hows and Whys of Early Steps in RNA Analysis, describes different methods for purifying RNA from fresh or fixed samples, protecting it from degradation and assessing quality before you proceed with downstream work. Register today to learn how you can achieve the best results possible with your RNA analysis studies.
DNA and RNA Purification Product Selector
Working with RNA (blog)
About the Webinar Series
www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition there are links to previous webinars that allow you to either view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.
To register for a webinar, use the “registration” link at: www.promega.com/webinars/ This allows you to view the webinar and participate in the live chat. Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page. Note: Live chat is only available for live webinars, not links to recorded webinars.
miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.
Consider the microRNA. At only about 21–26 nucleotides in length, microRNAs (miRNAs) are short, but don’t dismiss miRNAs as too short to accomplish much of anything. miRNAs are a multifunctional workhorse that play a key role in a number of genetic regulatory mechanisms throughout the plant and animal kingdoms and even in certain viruses. Scientists estimate that the human genome encodes about 2,000 different miRNAs and miRNAs account for about 3–4% of human genes (1).
Next Tuesday, we invite you to consider the microRNA with us as we host a webinar discussing the growing field of miRNA research and highlight a new, simplified miRNA purification method. Follow the link below to register.
Presented by Douglas Horejsh, Ph.D.
Tuesday, January 27
- Valinezhad Orang, A., Safaralizadeh, R. and Kazemzadeh-Bavili, M. (2014) Mechanisms of miRNA-mediated gene regulation from common downregulation to mRNA-specific upregulation. Int. J. Genomics Article ID 970607 http://dx.doi.org/10.1155/2014/970607
Formalin-fixed, paraffin-embedded (FFPE) tissue samples are extremely common sample types. In this form, tissue is easy to store for extremely long periods of time and useful for immunohistochemical studies. Additionally FFPE samples are fairly inexpensive to produce. However the formalin fixation procedure, which was developed long before the advent of molecular biology, results in chemical crosslinking of nucleic acid and protein molecules inside the cells. This crosslinking presents a challenge for isolating intact, high-quality nucleic acid DNA; so getting at the wealth of molecular information within an FFPE sample can be difficult.
In the upcoming webinar “Successfully Overcoming the Challenges of Working with FFPE Samples”, Dr. Trista Schagat of Promega Corporation discusses some of the key considerations for anyone who is attempting to isolate nucleic acid from FFPE samples. Continue reading
CellTox™ Green Dye Binds DNA Released By Dying Cells
If you have ever wondered about the differences between the various cell viability and cytotoxicity assays available, why you would choose one over another or how they can be used together, tune in to the webinar “A Real-Time Cytotoxicity Assay That Delivers More Relevant Data” (Tuesday, Sept 10). In this webinar, Promega Scientist Drew Niles explains how various metabolism and biomarker-based viability and cytotoxicity assays work, and describes how they can be used most effectively to give maximum information about mechanism and timing of cell death.
“Is a cell treatment toxic?” and “Why are the cells dying?” are questions that can be difficult to answer simply. The answer depends on dosage, treatment time, mechanism of action of the test compound, and the cell type used—and may sometimes be limited by features of the assay itself. For example, many viability and cytotoxicity assays measure biomarkers that are themselves subject to degradation over the course of longer experiments, complicating the interpretation of results. Drew provides an explanation of these issues and illustrates the critical role of timing in deciding on the assay to use and in interpreting results. Continue reading
This post written by guest blogger, Caroline Davis, Promega Technical Services Scientist.
Have you ever wished that you could have a scientist explain to you how to perform and optimize an experiment? Or do you want to learn more about your new area of research and wish that a scientist could present you with an overview of the field and discuss experimental approaches rather than having to do all of the background research yourself? Or are you interested in one of Promega’s instruments and would like to learn more about how to use the instrument and all of its features? If you answered yes to any of these questions, then a Promega Labinar may be right for you.
What is a Labinar?
A Promega Labinar is the next best thing to an in-person visit from one of our scientists. You simply tell us what you would like to learn about and a Promega Technical Service Scientist will prepare a presentation for you. The presentation will be live, not pre-recorded, and the level of detail will be tailored to meet your needs and experience.
How does it work?
You will need an internet connected computer or projection screen, as well as internet connected speakers or a speaker phone. Promega’s technical services department will send you instructions on how to “attend” the Labinar and test the video and audio connection with you ahead of time. There will be two-way audio so that you can comment, ask questions and fully participate in the seminar, just like an in-person seminar.
Examples of Previous Labinars Continue reading
Traditional techniques for studying protein:protein interactions (e.g., affinity capture or mass spectrometry) are limited in that they present a static picture of what is a dynamic process. To gain a more accurate and complete picture of interactions, they need to be studied n the context of the cellular environment within which they occur. In the webinar: Monitoring Protein:Protein Interactions in Living Cells Using NanoBRET™ Technology, Dr. Danette Daniels presented an improved Bioluminescence Resonance Energy Transfer (BRET) Technology that was developed to specifically study dynamic interactions in living cells. The NanoBRET™ Technology combines the small, extremely bright NanoLuc® Luciferase as the donor with a fluorescently labeled HaloTag® protein fusion tag as the acceptor to form a BRET assay. This assay offers an increased signal and decreased spectral overlap compared to other BRET technologies. Among other things, these features provide a larger signal range and the brightness of the NanoLuc® Luciferase enables protein:protein interaction analysis even with the donor protein expressed at low levels. Continue reading
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Considering automation for your laboratory? Wondering if there is a less time-intensive way to get through your workflow? Having problems purifying nucleic acids or protein from your samples or with your current protocol? Our recent webinar “Customized and Automated Solutions to Overcome Challenges in Your Lab” covered this topic, explaining there is team at Promega that can help. Promega Field Support Scientists (FSS) have the technical knowledge and automation expertise to develop customized solutions for laboratories that desire a customized protocol or automated workflow but do not have the time or understanding of liquid-handling workstations.
One point emphasized during the webinar was that our FSS team recommends that any assays or chemistries you are interested in automating are first tested and developed manually. Continue reading
When I was characterizing proteins in graduate school, my life was filled with constructs, constructs, constructs. I made a variety of subclones to synthesize and isolate parts and pieces of the protein in vitro. I made clones and subclones to generate a panel of antibodies against different parts of the protein. Some of those antibodies ended up working best on Westerns; others performed better in immunocytochemistry experiments. There was no one tool or tag that could be used for every step in the characterization of the protein.
HaloTag® Technology: A single, multifunctional protein fusion tag.
Halotag® fusion tag changes that.
The HaloTag® fusion tag spans both worlds of looking at proteins in isolation for studies of protein interactions and post-translational modifications to studying proteins in cells through real-time imaging or localization studies. Continue reading