Free-living amoebae (FLA), particularly Vermamoeba vermiformis and Acanthamoeba spp., have emerged as indispensable models for the study of giant viruses. These organisms provide a unique and efficient system for the isolation and replication of giant viruses, serving as known permissive hosts and thus playing a pivotal role in advancing research in this field. FLA can alternate between vegetative (trophozoite) and resistant (cyst) forms during their life cycle. Current methods for FLA storage and preservation face limitations, including prolonged excystment times and low rates of trophozoite viability. To address these challenges, these protocols provide instructions for the storage and preservation of Vermamoeba vermiformis at refrigeration temperatures, allowing feasible and rapid reactivation of cultures as an alternative to the more fastidious cyst-based storage methods. This method is also applicable to other FLA species, such as Acanthamoeba castellanii and Acanthamoeba polyphaga © 2025 Wiley Periodicals LLC. Basic Protocol 1: Vermamoeba vermiformis propagation Basic Protocol 2: Preparation of Vermamoeba vermiformis stocks for cold room storage Basic Protocol 3: Reactivation of cold-stored Vermamoeba vermiformis.
Effectively searching the scholarly literature is a fundamental academic skill. However, the process can be overwhelming due to the vast amount of available research and the complexity of academic databases. This overview article provides a practical guide to navigating the literature with confidence, outlining key strategies for identifying relevant sources, refining search queries, and troubleshooting common challenges. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.
An assay and protocol framework are provided for utilizing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) at the point-of-care for diagnosing and monitoring a hypothetical zoonotic viral outbreak in a resource-limited area. This manuscript utilizes a previously published decision tree algorithm to determine an appropriate molecular diagnostic point-of-care test that can effectively address the outbreak presented in the hypothetical case study. © 2025 Wiley Periodicals LLC.
Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3'-end labeled probes that were labeled in-house and 5'-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3'-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC. Basic Protocol: Biotin-based northern blotting.
The laboratory mouse has been described as a "miracle" model organism, providing a window by which we may gain an understanding of ourselves. Since the first recorded mouse experiment in 1664, the mouse has become the most used animal model in biomedical research. Mice are ideally suited as a model organism because of their small size, short gestation period, large litter size, and genetic similarity to humans. This article provides a broad overview of the laboratory mouse as a model organism and is intended for undergraduates and those new to working with mice. We delve into the history of the laboratory mouse and outline important terminology to accurately describe research mice. The types of laboratory mice available to researchers are reviewed, including outbred stocks, inbred strains, immunocompromised mice, and genetically engineered mice. The critical role mice have played in advancing knowledge in the areas of oncology, immunology, and pharmacology is highlighted by examining the significant contribution of mice to Nobel Prize winning research. International mouse mutagenesis programs and accurate phenotyping of mouse models are outlined. We also explain important considerations for working with mice, including animal ethics; the welfare principles of replacement, refinement, and reduction; and the choice of mouse model in experimental design. Finally, we present practical advice for maintaining a mouse colony, which involves adequate training of staff, the logistics of mouse housing, monitoring colony health, and breeding strategies. Useful resources for working with mice are also listed. The aim of this overview is to equip the reader with a broad appreciation of the enormous potential and some of the complexities of working with the laboratory mouse in a quest to improve human health. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR/Cas) are revolutionary tools for predictable and repeatable genome editing. In this article, the CRISPR/Cas9 system will be used to engineer the genome of the yeast Saccharomyces cerevisiae . As a model organism utilized across biological disciplines, efficient and tailorable methods for engineering the yeast genome are indispensable for a variety of research, educational, and commercial applications. The protocols described here incorporate a simple restriction-free cloning strategy and digestion-based screening method that can be readily and easily modified to suit user-designed experimental needs. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Recombinant pCAS plasmid preparation Basic Protocol 2 : Barcode/editing fragment assembly Basic Protocol 3 : Gene editing by yeast co-transformation Alternate Protocol : Competent yeast preparation and transformation by a lithium acetate/single-stranded carrier method
The laboratory notebook, whether in hardcopy or electronic (ELN) format, represents a true, chronological record of a scientist's bench work, incorporating the primary source of all materials and information related to the design, execution, and outcomes (data) for a specific experiment. The notebook content thus represents a precise, legible record of what was done, why, and by whom. It also describes the outcome of the experiment and how this is related to the initial hypothesis on which performing the experiment was based. As an archive, a laboratory notebook allows others previously unassociated with the work to understand what was done and, if necessary, independently recreate the experiments. A laboratory notebook and its associated records (e.g., computer printouts or HPLC traces) also represent the vital record of the conception date of an invention and its reduction to practice, providing a timely and necessary legal record to support and defend patent applications. © 2020 Wiley Periodicals LLC.
Stored biological materials should have minimal pre-analytical variations in order to provide researchers with high-quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK-8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors. Basic Protocol: Flash-freezing fresh tissue with aluminum platform Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen Alternate Protocol 2: Freezing fresh tissue with dry ice.
In order for the scientific enterprise to ensure equitable participation for all identities, the settings of professional research labs must cultivate an environment that is inclusive of all backgrounds. We explore here strategies to consider for research labs interested in cultivating inclusive environments. Investigators enacting inclusive strategies must understand the social context of the lab members and their reasons for engaging in science research. For this to be authentic, principal investigators should spend time exploring their own social positioning as well as the purpose of their professional engagement. We unpack the philosophies behind these constructs and provide specific suggestions to prepare individuals to fully engage in the practice of inclusive mentoring in science research labs. © 2020 Wiley Periodicals LLC.
Conferences play an important role in enabling trainees to develop and apply competencies in science, in communication, and in networking during biomedical PhD and postdoctoral training. This article offers guidelines for trainees on how to use conferences to initiate, sustain, and strengthen connections, including in virtual conference formats which could become the norm in the future. Additionally, it provides tips for expanding professional networks via broad mechanisms such as informational interviews. Recommendations in this manuscript are applicable to trainees pursuing diverse career paths in different STEM fields including education, scientific research, policy, advocacy, consulting, and communication. © 2020 Wiley Periodicals LLC.
For success in research careers, scientists must be able to communicate their research questions, findings, and significance to both expert and nonexpert audiences. Scientists commonly disseminate their research using specialized communication products such as research articles, grant proposals, poster presentations, and scientific talks. The style and content of these communication products differ from the language usage of the general public and can be difficult for nonexperts to follow and access. For this reason, it is important to tailor scientific communications to the intended audience in order to ensure that the communication product achieves its goals, especially when communicating with nonexpert audiences. This article presents a framework to increase access to research and science literacy. It addresses aspects of communication that scientists should consider when producing a scientific communication product: audience, purpose, format, and significance (research narrative). These factors are essential for understanding the communication scenario and goals, which provide guidance when tailoring research communications to different audiences. © 2020 by John Wiley & Sons, Inc.
This article provides an overview of foundational concepts of business strategy and business development that scientists can apply to starting and expanding their research programs. It covers topics including: defining a value proposition, identifying stakeholders, considering research gaps, strategic collaborations, responsible hiring, strategic planning, and time management. © 2020 by John Wiley & Sons, Inc.
Arabidopsis thaliana is a small plant of significant economic and agronomic importance. While Arabidopsis is not a crop plant, it is not so different in its fundamental properties. Arabidopsis is a member of the Brassicaceae family, which constitutes one of the world's most economically important plant groups. According to the United Nations, globally Brassicaceae crops are worth $31 billion, and the number is likely to increase since a number of related species within this family are underutilized edible varieties. Its small size is an advantage to researchers with limited space and funding; simply put, a smaller plant requires fewer resources. Arabidopsis has been studied most intensely for the last 40 years and officially became a model plant in the late 1990s. Since then, the community has developed genetic and genomic resources so numerous that the barrier to entry to studying Arabidopsis is relatively low. This article provides a primer to how Arabidopsis came to be a model organism and highlights essential techniques every Arabidopsis researcher should be aware of to advance the pace of discovery. © 2019 by John Wiley & Sons, Inc.
By implementing more transparent research practices, authors have the opportunity to stand out and showcase work that is more reproducible, easier to build upon, and more credible. Scientists gain by making work easier to share and maintain within their own laboratories, and the scientific community gains by making underlying data or research materials more available for confirmation or making new discoveries. The following protocol gives authors step‐by‐step instructions for using the free and open source Open Science Framework (OSF) to create a data management plan, preregister their study, use version control, share data and other research materials, or post a preprint for quick and easy dissemination. © 2019 by John Wiley & Sons, Inc.
Since its introduction as a laboratory organism 50 years ago, the nematode worm Caenorhabditis elegans has become one of the most widely used and versatile models for nearly all aspects of biological and genomic research. Many experiments in C. elegans begin with the generation and analysis of mutants that affect a specific biological process, so genetic techniques are the foundation of worm research. Many different aspects of biology are being studied in C. elegans , and three different recent Nobel Prizes have recognized six researchers working with worms. In addition, C. elegans was the first multicellular organism to have its genome sequenced, so many of the standard genomic methods have also been pioneered in C. elegans . In fact, many novel techniques and ideas are initially tested in C. elegans because of its versatility as a research organism. It is also appropriate for introducing undergraduate students to research, and some of its strengths and challenges for this purpose are discussed. © 2019 The Authors.
The understanding and application of clustered regularly interspaced short palindromic repeat (CRISPR) systems and CRISPR‐associated (Cas) nucleases have helped genome editing become a standard laboratory technique. CRISPR‐Cas nucleases are RNA‐programmed DNA‐cutting enzymes that facilitate the introduction of intentional sequence changes into the genomes of experimental cells and organisms. This overview provides a background for genome editing and CRISPR‐Cas nucleases, as well as an overview of the technology used to assess the outcomes of genome editing. © 2019 The Authors.
In science and engineering, there are a range of easy-to-access software applications for smart phones, tablets, and laptop and desktop computers that greatly simplify basic and advanced laboratory research activities. The available software programs include numerous mobile and web-friendly applications for electronic laboratory notebooks, project planning, solution calculations, and advanced scientific calculations and analysis, and even simple-to-use apps for your favorite scientific meetings. A typical collection of mobile-friendly applications is discussed here in the context of preparing, performing, and presenting your research. © 2019 by John Wiley & Sons, Inc.
Today's science is largely funded by taxpayer dollars, and because of this, scientists have a responsibility to ensure that their research is being effectively communicated back to taxpayers and to the policy makers who determine the distribution of those funds. The importance and impact of effective science communication is compounded when research is used to inform legislative action. Science impacts policy, and policy can impact science. However, the formal education of scientists does not usually include specific training on interacting with science policy. This article describes strategies for engaging with science policy-starting with simple and easy policy actions, and delving into more complex event planning and group organizing tasks. Whether advocating for evidence-based policies or policies that impact the scientific enterprise or STEM education, practicing skills pertinent to science policy can help you gain comfort in translating your experiences and experiments into lasting change.
This article describes common laboratory procedures that can reduce the risk of culture contamination (sepsis), collectively referred as "aseptic technique." Two major strategies for aseptic work are described: using a Bunsen burner and using a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microorganisms on petri dishes. © 2020 by John Wiley & Sons, Inc.
This unit lists recipes for many of the buffers and other reagents typically found in a molecular biology laboratory. For reagents specific to a given technique, refer to the unit describing the method. © 2018 by John Wiley & Sons, Inc.
