Molecular Biology

This protocol presents a modified version of the Filterprep method originally reported in New Biotechnology, adding an optional step to reduce endotoxin levels. Filterprep is a simple, rapid, and cost-effective approach to plasmid DNA purification that couples ethanol precipitation with a single spin-column filtration step, eliminating chaotropic salts and silica binding. The formulations and parameters are fully transparent and do not rely on proprietary buffers, using only standard laboratory reagents and widely available miniprep columns. Under matched conditions, the method recovers high-purity plasmid DNA with yields up to fivefold higher than those obtained with representative commercial midiprep kits. The workflow is readily adoptable in most molecular biology laboratories and, under routine conditions, can be completed in approximately 40 min. The resulting DNA is suitable for molecular cloning, PCR, sequencing, and other downstream biochemical applications. Endotoxin is a lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria and may carry over during plasmid preparation. For experiments requiring lower endotoxin input, an optional modification resuspends the DNA pellet in a Triton X-114 wash buffer before column loading to decrease lipopolysaccharide carryover. The method is modular and extensible, allowing adjustment of precipitation and wash conditions, variation in the number of washes, selection of alternative column formats, and integration of endotoxin-reduction modules without altering the core principle. These features facilitate troubleshooting and quality control, enable scaling from routine batches to larger culture volumes and higher throughput, and allow seamless integration with existing workflows.

The exploration of microbial genomes through next-generation sequencing (NGS) and genome mining has transformed the discovery of natural products, revealing an immense reservoir of previously untapped chemical diversity. Bacteria remain a prolific source of specialized metabolites with potential applications in medicine and biotechnology. Here, we present a protocol to access novel biosynthetic gene clusters (BGCs) that encode natural products from soil bacteria. The protocol uses a combination of Oxford Nanopore Technology (ONT) sequencing, de novo genome assembly, antiSMASH for BGC identification, and transformation-associated recombination (TAR) for cloning the BGCs. We used this protocol to allow the detection of large BGCs at a relatively fast and low-cost DNA sequencing. The protocol can be applied to diverse bacteria, provided that sufficient high-molecular-weight DNA can be obtained for long-read sequencing. Moreover, this protocol enables subsequent cloning of uncharacterized BGCs into a genome engineering-ready vector, illustrating the capabilities of this powerful and cost-effective strategy.

A simple and effective method to identify genetic markers of yield response to nitrogen (N) fertilizer among maize hybrids is urgently needed. In this article, we describe a detailed methodology to identify genetic markers and develop associated assays for the prediction of yield N-response in maize. We first outline an in silico workflow to identify high-priority single-nucleotide polymorphism (SNP) markers from genome-wide association studies (GWAS). We then describe a detailed methodology to develop cleaved amplified polymorphic sequences (CAPS) and derived CAPS (dCAPS)-based assays to quickly and effectively test genetic marker subsets. This protocol is expected to provide a robust approach to determine N-response type among maize germplasm, including elite commercial varieties, allowing more appropriate on-farm N application rates, minimizing N fertilizer waste.

Zebrafish are a powerful model for investigating vascular and lymphatic biology due to their genetic tractability and optical transparency. While translating ribosome affinity purification (TRAP) has been widely applied in other systems, its application in zebrafish has remained limited. Here, we present an optimized TRAP protocol for isolating ribosome-associated mRNAs from endothelial cells in vivo, without the need for cell dissociation or sorting. Using a novel transgenic zebrafish line, which expresses HA-tagged Rpl10a under the mrc1a promoter, we enriched actively translating endothelial transcripts. Differential expression analysis revealed robust upregulation of vascular and lymphatic genes including flt4, kdrl, and lyve1b. This approach captures the endothelial cell translatome with high specificity and offers a robust platform for investigating the molecular mechanisms of endothelial biology under genetic, environmental, or toxicological perturbations.

Genome-walking protocols have been extensively used to clone unknown genomic sequences next to known DNAs. Existing genome-walking protocols need further improvement in methodological specificity or operation. Here, we describe a novel genome-walking protocol based on fusion primer–driven racket PCR (FPR-PCR). FPR-PCR involves four sequence-specific oligos (SSO), SSO1, SSO2, SSO3, and SSO4, which are sequentially chosen from known DNA in the direction 5’→3’. The fusion primer, mediating primary FPR-PCR, is generated by attaching SSO3 to the 5’ end of SSO1. The SSO3 encourages the target DNA of primary PCR to form a racket-like structure by mediating intra-strand annealing. SSO2 and SSO4 are directly used as sequence-specific primers (SSP) in secondary FPR-PCR, which selectively amplifies this racket-like DNA. This protocol was verified by cloning several unknown genomic sequences. Compared to traditional PCRs, FPR-PCR offers the advantages of higher specificity and fewer rounds, primarily attributed to the omission of arbitrary walking primers typically required in traditional methods.

Genome walking is a classical molecular biology technique used to amplify unknown regions flanking known DNA sequences. Genome walking holds a vital position in the areas associated with molecular biology. However, existing genome-walking protocols still face issues in experimental operation or methodological specificity. Here, we propose a novel genome-walking protocol based on bridging PCR. The critical factor of this protocol is the use of a bridging primer, which is made by attaching an oligomer (or tail primer sequence) to the 5′ end of the walker primer 5′ region. When the bridging primer anneals to the walker primer site, this site will elongate along the tail of the bridging primer. The non-target product (the main contributor to background in genome walking), defined by the walker primer, is lengthened at both ends. In the next PCR(s), the annealing between the two lengthened ends is easier than the annealing between them and the shorter tail primer. As a result, this non-target product is eliminated without affecting target amplification.

Protein phosphorylation is a dynamic post-translational modification that regulates fundamental processes, including signal transduction, cell proliferation, differentiation, and effector function of immune cells. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is a key mediator of cytokine responses, essential for maintaining immune cell homeostasis and determining cell fate across diverse immune subsets. Dysregulation of JAK/STAT signaling has been linked to a broad spectrum of pathologies, including monogenic immune disorders, autoimmunity, and cancer. Platforms facilitating single-cell analysis of protein phosphorylation offer the ability to reveal subtle signaling defects and dissect the pleiotropy in cellular composition and phosphorylation status, providing insights into immune phenotype and function, while identifying potential therapeutic targets. While an application of cytometry-by-time-of-flight, termed phospho-CyTOF, has proven invaluable for studying protein phosphorylation in cryopreserved peripheral blood mononuclear cells (cPBMCs), its application is limited by cell loss and signaling artifacts stemming from isolation and cryopreservation. Conversely, whole blood (WB) approaches, preserving the native immune cell composition and signaling context, offer a more physiological representation but necessitate robust and consistent protocols for broad application. Herein, we present optimized dual phospho-CyTOF workflows tailored for both cPBMCs and whole blood, building upon established protocols for cytokine stimulation of both samples. These workflows facilitate comprehensive, high-dimensional profiling of JAK/STAT signaling in response to pleiotropic cytokines such as Type I interferons (IFN-α), Type II interferons (IFN-γ), and Interleukin-21 (IL-21). By leveraging CyTOF's capacity for high-dimensional profiling using pure heavy metal–labeled antibodies, these protocols aim to identify pathway-specific alterations in STAT phosphorylation across major immune subsets that may be overlooked by traditional flow cytometry. Together, these optimized dual workflows provide scalable, translationally relevant tools for dissecting the subtle and differential JAK/STAT-driven immune responses in both clinical and research settings, while also being compatible with the simultaneous assessment of crosstalk with alternative immune cell signaling pathways.

Real-time quantitative PCR (qPCR) is a pivotal technique for analyzing gene expression and DNA copy number variations. However, the limited availability of user-friendly software tools for qPCR data analysis presents a significant challenge for experimental biologists with limited computational skills. To address this issue, we developed Click-qPCR, a user-friendly and web-based Shiny application for qPCR data analysis. Click-qPCR streamlines ΔCq and ΔΔCq calculations using user-uploaded CSV data files. The interactive interface of the application allows users to select genes and experimental groups and perform Welch’s t tests and one-way analysis of variance with Dunnett’s post-hoc test for pairwise and multi-group comparisons, respectively. Results are visualized via interactive bar plots (mean ± standard deviation with individual data points) and can be downloaded as publication-quality images, along with summary statistics. Click-qPCR empowers researchers to efficiently process, interpret, and visualize qPCR data regardless of their programming experience, thereby facilitating routine analysis tasks. Click-qPCR Shiny application is available at https://kubo-azu.shinyapps.io/Click-qPCR/, while its source code and user guide are available at https://github.com/kubo-azu/Click-qPCR.

Preserving biological samples in the field is essential for ensuring high-quality nucleic acid extraction and reliable downstream molecular analyses. Broadly, two main preservation strategies are available: physical preservation, such as flash freezing in liquid nitrogen, which halts enzymatic activity by rapid cooling, and chemical preservation, using stabilizing reagents that inactivate nucleases and protect nucleic acids even at ambient temperatures. This protocol presents a comparative approach using liquid nitrogen and a commercial stabilizing reagent (DNA/RNA Shield, Zymo Research) to preserve tissue from five marine invertebrate species: two cold-water corals, two sponges, and one bivalve. Samples preserved by each method were processed with the AllPrep DNA/RNA Mini kit (Qiagen) to extract both RNA and DNA. RNA quality was assessed using RNA Integrity Number (RIN) scores. The stabilizing reagent preserved high-quality RNA in sponge and bivalve samples but did not prevent RNA degradation in coral tissues, which showed lower RIN scores compared to those preserved in liquid nitrogen. DNA yields were also consistently lower in tissues preserved with DNA/RNA Shield across all species. These findings suggest that DNA/RNA Shield can be a viable alternative to liquid nitrogen for some marine invertebrates, particularly in field conditions where cryopreservation is impractical. However, for cold-water corals, liquid nitrogen remains essential to ensure RNA integrity for transcriptomic analyses and other sensitive molecular applications (e.g., RT-qPCR).

Telomere length maintenance is strongly linked to cellular aging, as telomeres progressively shorten with each cell division. This phenomenon is well-documented in mitotic, or dividing, cells. However, neurons are post-mitotic and do not undergo mitosis, meaning they lack the classical mechanisms through which telomere shortening occurs. Despite this, neurons retain telomeres that protect chromosomal ends. The role of telomeres in neurons has gained interest, particularly in the context of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where aging is a major risk factor. This has sparked interest in investigating telomere maintenance mechanisms in post-mitotic neurons. Nevertheless, most existing telomere analysis techniques were developed for and optimized using mitotic cells, posing challenges for studying telomeres in non-dividing neuronal cells. Thus, this protocol adapts an already established technique, the combined immunofluorescence and telomere fluorescent in situ hybridization (IF-FISH) on mitotic cells to study the processes occurring at telomeres in cortical neurons of the mouse ALS transgenic model, TDP-43 rNLS. Specifically, it determines the occurrence of DNA damage and the alternative lengthening of telomeres (ALT) mechanism through simultaneous labeling of the DNA damage marker, γH2AX, or the ALT marker, promyelocytic leukemia (PML) protein, together with telomeres. Therefore, the protocol enables the visualization of DNA damage (γH2AX) or the ALT marker (PML) concurrently with telomeres. This technique can be successfully applied to brain tissue and enables the investigation of telomeres specifically in cortical neurons, rather than in bulk tissue, offering a significant advantage over Southern blot or qPCR-based techniques.