Current Protocols

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@417649org.highwire.dtl.DTLVardef@1bcd186org.highwire.dtl.DTLVardef@15fecb3org.highwire.dtl.DTLVardef@acdf8d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during the extraction process. We tested whether using an alternative Plant kit RNA extraction method to wash agarose impurities or pre-dissolving agarose from PCLS implementing a dissolving buffer for routine RNA isolation in gel-electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The plant kit and dissolution of agarose increased the RNA quantity to 0.42{+/-}0.11 and 0.65{+/-}0.17 {micro}g/PCLS (measured by the Qubit) and integrity number to 6.60{+/-}0.59 and 9.13{+/-}0.39 (measured by the Bioanalyzer), respectively. The presence of impurities in conventional and Plant kit extractions misled to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcript integrity increase in GUSB (p<0.0001) and COL1A1 (p<0.05) expression, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods to remove agarose impurities during RNA extraction, as well as to rely on sensitive quantitative techniques, such as the Qubit and Bioanalyzer, for RNA quantification and integrity measurements.

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% (p-value < 0.0005) "sense" to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.

Winnie mice are a widely used in vivo model of inflammatory bowel disease carrying a missense mutation in the Muc2 gene. Here, we present a protocol for genotyping Winnie mice using TaqMan allelic discrimination quantitative PCR. We describe tissue collection, rapid crude DNA extraction, probe-based amplification with dual-labeled fluorophores, and fluorescence-based genotype calling in a single reaction. This protocol enables qualitative SNP genotyping without post-amplification processing and can be readily adapted to other defined point mutations. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/704640v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f5d985org.highwire.dtl.DTLVardef@19bbd34org.highwire.dtl.DTLVardef@1a2d2fcorg.highwire.dtl.DTLVardef@c9baed_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAllelic discrimination qPCR protocol for genotyping the Muc2 p.Cys52Tyr mutation using dual-labeled hydrolysis probes C_LIO_LIEnables rapid discrimination of wild-type, heterozygous, and mutant alleles in a single reaction C_LIO_LICompatible with standard real-time PCR instruments and requires no post-PCR processing C_LIO_LISupports high-throughput genotyping from crude DNA with minimal hands-on time C_LI

Low take rates and inter-tumor variability in growth rates can limit the effectiveness of mouse xenograft models when comparing between groups. To address this problem we developed a simple method to compare multiple cell types within a single mixed xenograft. Individual cell lines or clones were transduced with a lentiviral vector that includes a unique PCR tag, allowing the use of qPCR to determine the proportion of each tagged cell type within a mixed xenograft tumor. We generated vectors with six distinct PCR tags, and two different selectable markers, and have optimized the approach for determining their relative proportions within a mix. An initial pre-amplification step is used to increase the amount of material for subsequent qPCR reactions. This also removes the bulk of the genomic DNA, increasing the specificity of the qPCR step. Samples are then used for qPCR with specific pairs of primers that distinguish between each of the individual PCR tags, and the relative proportion of each tag is determined relative to that in the starting mix. We have tested this approach for in vitro growth of mixed cell cultures and in an orthotopic cecal xenograft model using a human colon cancer cell line. Since each individual tumor is initiated with a mix of cells, multiple tumors within a single animal can be analyzed separately, and overall tumor size is not important. Similarly, multiple metastatic lesions from the same animal can be analyzed individually. Thus, each tumor provides a direct comparison between individually tagged cell lines or clones. This low throughput "bar-coding" approach is simple and cost effective and has the potential to reduce the number of animals needed for xenograft experiments.

Transgenic mouse lines are essential to uncover organ or system level genotype-phenotype relationships. The generation of such lines via transgene addition may lead to the insertion into unknown genomic loci potentially leading not only to the disruption of native genes but also attenuation of transgene expression. Additionally, this often results in the inability to determine transgene zygosity which in turn complicates breeding and interpretation of experimental results. In this study we present two whole genome sequencing based pipelines that allow the identification and genotyping of even complex multi transgenic inserts. As they use widely available reagents and bioinformatic tools, they can easily be applied to develop genotyping strategies in potentially any species.

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing because myelin and debris can reduce nuclei quality. We describe an optimized protocol for mouse hippocampi and cerebella using tube-and-pestle homogenization and low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, with optional magnetic enrichment of nuclei to reduce debris/non-nuclear carryover. Under the tested conditions, the workflow produces intact, debris-reduced nuclei and supports downstream 10x Genomics Flex and PARSE WT library preparation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/716374v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ccbd87org.highwire.dtl.DTLVardef@1aef4bcorg.highwire.dtl.DTLVardef@14569a8org.highwire.dtl.DTLVardef@1bc261_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBenchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brain C_LIO_LIScales across tissue inputs (e.g., hippocampus [~]15-20 mg; cerebellum [~]50-70 mg) without ultracentrifugation or 15 mL gradients C_LIO_LIMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows. C_LI

Transposon insertion sequencing (TIS) encompasses methods such as Transposon Directed Insertion sequencing (TraDIS) and Transposon-Sequencing (Tn-Seq); these methods are widely uses for genome scale screens of essential and conditionally essential genes. Early limitations centred on mutant generation, today the biggest factor is sequencing and data generation and the costs associated. Here, we present and evaluate four methods TIS sequencing library generation protocols compatible with Illumina whole genome sequencing (WGS) workflows to maximise sequence efficiency and minimise turnaround times and costs. Using E.coli BW25113 transposon mutagenesis libraries generated with Tn5 and mariner (Himar1) transposons, we compare the methods in terms of reagent cost, workflow complexity, and target enrichments for the recovery of unique insertion sites and essential genes identified. All methods generated TIS data suitable for gene essentiality analysis. Illumina Flex based protocols were 4-6 tomes cheaper than the traditional Illumina Nextera based approach with similar or superior Transposon-Chromosome (Tn-Chr) junction enrichment. Across all methods, sequencing depth and mutant library density were the dominant factor in useful biological insight. Subsampling demonstrated that for a good quality mutant library, five million reads were sufficient to identify essential genes in E.coli. Whereas deeper sequencing reduced the statistical power and included contaminating background noise, seen primarily with Tn5. We conclude that an Illumina Flex based approach, especially when integrating with routine WGS, provides an excellent balance of speed, cost and data quality. Assuming five million reads and a robust Illumina Flex approach, a TIS library can be sequenced for around {pound}40.

The coleoid cephalopods (octopus, cuttlefish, and squid) are emerging model organisms for neuroscience, development, and evolutionary biology. Determining their sex early in life is critical for population management and controlled experiments. Here, we present a protocol to non-invasively determine the sex of multiple cephalopod species as young as 3 hours post-hatching using a skin swab and quantitative PCR (qPCR). We describe steps for designing qPCR primers, swabbing live animals, extracting DNA, running the qPCR, and analyzing the results. For complete details on the use and execution of this protocol, please refer to Rubino et al.1 HighlightsO_LISwab live cephalopods as early as 3 hours post-hatching C_LIO_LIExtract DNA from cephalopod skin swabs C_LIO_LIPerform qPCR-based sex determination C_LIO_LIDesign and validate qPCR primers for new species C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/715692v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3aa68dorg.highwire.dtl.DTLVardef@8c7e61org.highwire.dtl.DTLVardef@1bd45d9org.highwire.dtl.DTLVardef@134cc4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Traditional nucleic acid extraction methods are costly, lengthy, and highly variable depending on the complexity of the sample matrix or the organism of interest. Workflows may exceed twenty steps, require separate kits for RNA and DNA, and demand expensive instrumentation, creating barriers to both speed and scalability. The AutolabTM HBH system addresses these limitations by using hyperbaric heating (HBH) to achieve temperatures above 100 {degrees}C in a sealed, pressurized environment through induction heating, enabling rapid lysis of diverse organisms and neutralization of macromolecular PCR inhibitors within minutes. The combination of extreme heat and HBH-optimized lyophilized reagents rapidly inactivates nucleases while preserving free nucleic acids. The workflow is streamlined to two steps: heating up to 1 mL of sample in the proprietary HBH bullet, followed by a brief centrifugation to pellet additives. The resulting supernatant is immediately compatible with real-time reverse transcription polymerase chain reaction (RT-PCR) and other downstream molecular assays. Here, we evaluate the systems broad compatibility with diverse sample buffers, matrices, and organisms. Comparative testing was conducted alongside Qiagen extraction methods to benchmark performance.

IntroductionImproving the efficiency and accuracy of annotation and extraction of performance data from mouse behavioural tasks will improve both the throughput and scientific value of preclinical research. MethodsHere, we present and validate an automated pipeline for the annotation and quantification of performance in a mouse olfactory habituation-dishabituation task, using a single side-view camera, resulting in occluded body parts. We created a pipeline for task analysis, combining DeepLabCut, for pose-estimation, and SimBA, for behavioural classification to automatically quantify odour interaction (sniffing time) in a three-odour (water, familiar mouse social odour, novel mouse social odour) variant of the task. We used a subset of previously published, fully manually annotated datasets to train the models and unseen videos from the same study to validate the utility of our machine learning pipeline. Results and conclusionOur analysis pipeline estimated behavioural performance in the task with high accuracy, and the data produces similar technical and biological results to manual methods when analysed by linear mixed modelling. Thus, we validated the utility of our new pipeline for the automated scoring of this mouse sensory task.

Antisense vivo-morpholino oligonucleotides (vivo-MOs) allow transient gene knockdown in adult organisms with high specificity and low toxicity. Vivo-MOs are used in cell culture and in many established model organisms, but a method for their use has not been described in threepsine stickleback (Gasterosteus aculeatus (Linnaeus, 1758)). Stickleback are an emerging model system used in evolutionary and ecological genetic studies. While genomic techniques are commonly used in stickleback research, there are few studies and tools available to assess gene function in-vivo, especially for genes that may be difficult to knock out by CRISPR (e.g., lethal knock-outs). Here, we test the use of splice-blocking vivo-MOs for gene knockdown in stickleback using intraperitoneal injection of vivo-MOs targeting three candidate genes. Gene expression was assessed in the liver, spleen, and intestine. Successful knockdown of Spi1b was observed in the spleen, however, we observed no other significant knockdown at either timepoint tested. Injection of a fluorescently labeled control vivo-MO confirmed delivery to each target organ, validating this approach, but delivery was variable which may explain inconsistent effects. These results indicate that vivo-MOs have potential as a tool for in-vivo gene knockdown in stickleback. Optimizing delivery methods could improve reproducibility and knockdown efficiency in future studies.

Octopus vulgaris and other cephalopods are of increasing interest as neurobiological model organisms. This protocol describes a method to record calcium activity from individual cells in acute brain slices from Octopus vulgaris hatchlings during exogenous application of neurotransmitters. Using this protocol, we characterized single-cell responses to specific neurotransmitters in the optic lobes, which process visual information. The approach is readily adaptable to other cephalopods and small invertebrate species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/711860v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1564eaeorg.highwire.dtl.DTLVardef@147b682org.highwire.dtl.DTLVardef@11f3b85org.highwire.dtl.DTLVardef@17c9d70_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cryopreservation, or cryonics, is an experimental procedure that preserves individuals at cryogenic temperatures after legal death in the hope of future revival. Although Switzerland hosts Schengen Areas first dedicated cryopreservation facility, public sentiment toward the practice has remained largely unexamined. This exploratory survey of 249 Swiss adults assessed awareness, ethical views, and openness to cryopreservation. Results show broad support for individual autonomy, with most respondents endorsing the right to choose cryopreservation when performed to high medical standards (86.7%) and not supporting legal restrictions (83.5%). While personal interest was in the minority, nearly one in five respondents (20.1%) reported active interest or intent to sign up. Openness to cryopreservation appears driven more by values such as life-extension preference and prior exposure than by demographics. These findings provide the first empirical snapshot of Swiss public opinion on cryopreservation, highlighting a largely permissive public stance and suggesting considerable engagement with the topic.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

Quantitative histological analysis of skeletal muscle morphometry provides critical insights into muscle physiology but remains labor-intensive and technically demanding. While recent developments in machine-learning-based image segmentation techniques have facilitated large-scale tissue analysis, existing tools that automate muscle morphometry analysis are largely tailored to mammalian models, with limited applicability to teleosts. Moreover, there is a lack of effective tools for visualizing spatial organization and morphometric variability of teleost muscle fibers, a feature that is important for understanding hyperplastic muscle growth dynamics in teleosts. In this study, we show that cytoplasmic staining combined with deep learning-based cell segmentation offers a robust and accurate approach for automated muscle morphometry analysis in developing zebrafish. We also introduce a FIJI2 plugin, implemented in Jython, that streamlines both morphometric analysis and visualization. This tool accommodates shallow and deep learning-based segmentation techniques and incorporates novel quantification and visualization methods suited to teleost-specific muscle features, including mosaic hyperplasia dynamics. The plugin features an intuitive graphical user interface and is designed for flexibility, with minimal constraints regarding species, image quality, or staining protocol. Its modular architecture allows it to be used as a baseline for automated muscle morphometry analysis, while permitting integration with other tools and workflows.

Computational bioacoustics has seen significant advances in recent decades. However, the rate of insights from automated analysis of bioacoustic audio lags behind our rate of collecting the data - due to key capacity constraints in data annotation and bioacoustic algorithm development. Gaps in analysis methodology persist: not because they are intractable, but because of resource limitations in the bioacoustics community. To bridge these gaps, we advocate the open science method of data challenges, structured as public contests. We conducted a bioacoustics data challenge named BioDCASE, within the format of an existing event (DCASE). In this work we report on the procedures needed to select and then conduct useful bioacoustics data challenges. We consider aspects of task design such as dataset curation, annotation, and evaluation metrics. We report the three tasks included in BioDCASE 2025 and the resulting progress made. Based on this we make recommendations for open community initiatives in computational bioacoustics.

MOTIVATIONLongitudinal molecular studies of the mouse brain are limited by the need for terminal tissue collection. This prevents analysis of preexisting molecular states and their evolution within the same individual. We developed a stereotactic microbiopsy technique that enables minimally invasive sampling of defined brain regions in vivo. The method preserves survival while yielding material suitable for RNA and nuclei isolation. It provides a practical solution for linking baseline molecular states to subsequent behavioural, pharmacological, or disease-related outcomes. SUMMARYThis study presents a stereotactic microbiopsy technique for sampling defined brain regions in living mice, enabling transcriptomic and epigenomic analyses without sacrificing the animal. The method will allow pre-intervention tissue collection, making it possible to separate preexisting molecular differences from experience- or treatment-induced changes. We show that microbiopsies yield sufficient, high-quality RNA and chromatin for sequencing, with minimal tissue damage that largely resolves over time. The procedure uses standard stereotactic equipment and achieves reproducible spatial precision when the syringe is stabilised. This approach provides a practical framework for within-subject molecular comparisons, reducing animal use and enabling longitudinal profiling of the living mouse brain. It establishes a foundation for investigating how baseline molecular states influence later physiological or behavioural outcomes.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.