dna

Why mRNA Transfection Is Transforming Transient Expression Workflows

Posted on by Shannon Sindermann

Transfection is a core technique in molecular biology used to introduce foreign nucleic acids—such as DNA, RNA, or small RNAs like siRNA, shRNA, and miRNA—into eukaryotic cells. This enables researchers to manipulate gene expression and study cellular processes, disease mechanisms and therapeutic strategies (1).

Advances in transfection technology now support a range of nucleic acid types and cell models. Researchers can pursue transient or stable expression to achieve specific goals: knocking down transcripts, expressing proteins, or probing promoter activity in systems from immortalized lines to stem cells (1).

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5 Questions to Ask When Your RT-qPCR Isn’t Working

Posted on by Shannon Sindermann
RT-qPCR

RT-qPCR (reverse transcription quantitative PCR) is a powerful technique for quantifying RNA expression—but it doesn’t always cooperate. Even when you’ve followed the protocol carefully, unexpected results can appear: flat curves, unexpected Cq values, or inconsistent replicates. When that happens, you’re left wondering… what went wrong?

In this blog, we’ll walk through five key questions to help you troubleshoot RT-qPCR issues with confidence. From common errors to more stubborn challenges, we’ll also explore what to consider when technique isn’t fully the problem—and when it might be time to rethink your reagents.

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Overcoming qPCR Inhibitors: Strategies for Reliable Quantification 

Posted on by Promega

Today’s blog is written by guest blogger, Gabriela Saldanha, Senior Product Marketing Manager at Promega.

Quantitative PCR (qPCR) is an indispensable tool for nucleic acid analysis, widely used in research, clinical diagnostics and applied sciences. Its sensitivity and specificity make it a powerful method for detecting and quantifying DNA and RNA targets. However, qPCR reactions are highly susceptible to inhibitors—substances that interfere with enzyme activity, primer binding, or fluorescent signal detection. These inhibitors can originate from biological samples, environmental contaminants, or laboratory reagents, potentially leading to inaccurate quantification, poor amplification efficiency, or complete reaction failure.

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No Horsin’ around with Halal Meat Authentication

Posted on by Promega

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.  

People’s diets are frequently influenced by a wide range of variables; with environment, socioeconomic status, religion, and culture being a few of the key influencers. The Muslim community serves as one illustration of how culture and religion can hold influence over people’s eating habits.

Halal meat on cutting board

Muslims, who adhere to Islamic teachings derived from the Qur’an, frequently base dietary choices on a food’s halal status, whether it is permissible to consume, or haram status, forbidden to consume. With the population of Muslims expected to expand from 1.6 billion in 2010 to 2.2 billion by 2030, the demand for halal products is anticipated to surge (2).

By 2030, the global halal meat market is projected to reach over $300 billion dollars, with Asia-Pacific and the Middle East regions being the largest consumers and producers of halal meat products (3). Furthermore, increasing awareness and popularity of halal meat among non-Muslim consumers, as well as strengthening preference for ethical and high-quality meat, are all contributing to demand.  

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In Vitro Transcription and the Use of Modified Nucleotides

Posted on by Leah Cronan
In vitro transcription
RNA polymerase unwinds DNA strands for transcription.

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.

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Reliable DNA Purification from 3D Cell Cultures

Posted on by Julia Nepper

Traditionally, scientists have relied on flat, two-dimensional cell cultures grown on substrates such as tissue culture polystyrene (TCPS) to study cellular physiology. These models are simple and cost-effective to culture and process. Within the last decade, however, three-dimensional (3D) cell cultures have become increasingly popular because they are more physiologically relevant and better represent in vivo conditions.

A spheroid of ~1,000 human liver cells. Image provided by Insphero.
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Moving Towards Zero Hunger, One Genome at a Time

Posted on by Julia Nepper
Farmer and a pile of cassava bulbs.

Have you ever thought about plant viruses? Unless you’re a farmer or avid gardener, probably not. And yet, for many people the battle against agricultural viruses never ends. Plant viruses cause billions of dollars in damage every year and leave millions of people food insecure (1–2), making viruses a major barrier to meeting the United Nations’ global sustainable development goal of Zero Hunger by 2030.

At the University of Western Australia, Senior Research Fellow Dr. Laura Boykin is using genomics and supercomputing to tackle the problem of viral plant diseases. In a recent study, Dr. Boykin and her colleagues used genome sequencing to inform disease management in cassava crops. For this work, they used the MinION, a miniature, portable sequencer made by Oxford Nanopore Technologies, to fully sequence the genomes of viruses infecting cassava plants.

Cassava (Manihot esculenta) is one of the 5 most important calorie sources worldwide (3). Over 800 million people rely on cassava for food and/or income (4). Cassava is susceptible to a group of viruses called begomoviruses, which are transmitted by whiteflies. Resistant cassava varieties are available. However, these resistant plants are usually only protected against a small number of begomoviruses, so proper deployment of these plants means farmers must know both whether their plants are infected and, if so, the strain of virus that’s causing the infection.

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Of Mice and Microbes: The Science Behind Food Analysis

Posted on by Julia Nepper

In general, people like to know that their food is what the label says it is. It’s a real bummer to find out that beef lasagna you just ate was actually horsemeat. Plus, there are many religious, ethical and medical reasons to be cognizant of what you eat. Someone who’s gluten intolerant and Halal probably doesn’t want a bite of that BLT.

Labels don’t always accurately reflect what is in food. So how do we confirm that we are in fact buying crab, and not whitefish with a side of Vibrio contamination?

For the most part, it comes down to separation science. Scientists and technicians use various chromatographic methods, such as gas chromatography, liquid chromatography, and mass spectrometry, to separate the complex mixture of molecules in food into individual components. By first mapping out the molecular profile of reference samples, they can then take an unknown sample and compare its profile to what it should look like. If the two don’t match up, an analyst would assume that the unknown is not what it claims to be. Continue reading “Of Mice and Microbes: The Science Behind Food Analysis”

High-Throughput Purification with Experts Included

Posted on by Darcia Schweitzer

Implementing automated nucleic acid purification or making changes to your high-throughput (HT) workflow can be complicated and time-consuming. There are also many barriers to success such as challenging samples types and maintaining desirable downstream results that can add to the stress, not to mention actually getting the robotic instrumentation to do what you want it to. All of this makes it easy to understand why many labs avoid automating or own expensive instrumentation that goes unused. Continue reading “High-Throughput Purification with Experts Included”

Better NGS Size Selection

Posted on by Darcia Schweitzer

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.

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